Drug delivery system for hydrophobic drugs

ABSTRACT

Compositions comprising microaggregates containing hydrophobic drugs, as well as methods for their production, are described. Such microaggregates may include micelle structures or combinations thereof with liposomes, and constitute an effective delivery vehicle for a hydrophobic agent. Methods for microaggregate production include the use of preferred lipid compounds and processing conditions favoring the production of small aggregates for improved filter sterilization.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/254,400 filed 20Oct. 2005, which is a continuation of U.S. Ser. No. 09/833,406 filed 11Apr. 2001, now U.S. Pat. No. 6,984,395. The contents of these documentsare incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to water soluble microaggregates of waterinsoluble, poorly soluble or otherwise hydrophobic agents andphospholipids or lipids which may be used pharmaceutically,agriculturally or industrially. These microaggregate compositions may beused to deliver hydrophobic drugs as a pharmaceutical formulation,hydrophobic compounds related to plant growth as an agriculturalproduct, and hydrophobic reagents as an industrial material. Moreover,the microaggregates of the invention comprise combinations of naturaland/or synthetic phospholipids which permit aggregation with thehydrophobic agents to result in micelles, liposomes, and mixturesthereof. Particular combinations of hydrophobic agents and phospholipidsor lipids produce microaggregates that are effective delivery vehiclesof said compounds.

Additionally, the invention relates to processes for the production ofsaid microaggregates as delivery systems. These processes includemicrofluidization (liquid jet milling), high shear mixing, andsonication. Particular processes, involving the use of specificcombinations of hydrophobic agents and phospholipids or lipids, permitthe large scale preparation of effective delivery vehicles forhydrophobic agents.

DESCRIPTION OF THE RELATED ART

The existence of a wide array of active hydrophobic or otherwise waterinsoluble agents is known in the art. Similarly there is awareness ofthe need to deliver such active agents to water based or otherwiseaqueous environments. As such, multiple systems have been development asdelivery vehicles for such agents. These include the use of organicsolvents, aqueous/detergent mixtures, aqueous/organic solvent mixtures(such as co-solvents), emulsions, liposomes, and micelles. Each of thesesystems, however, have limitations arising from considerations such asthe degree of water insolubility and the environment into which deliveryis desired.

An example of hydrophobic agents in liposomes is taught by Farmer etal., U.S. Pat. No. 4,776,991, which discloses the large-scaleencapsulation of hemoglobin. Kappas et al., U.S. Pat. No. 5,010,073,discloses the preparation of liposomes containing a metalloporphyrinwith egg phosphatidyl choline (“EPC”) being used as the lipid. Schneideret al., U.S. Pat. No. 5,270,053, discloses liposome formulations said tobe free of solid particles and larger lipid aggregates. Parikh et al.,U.S. Pat. No. 5,922,355, disclose microparticles comprising insolublesubstances. Lasic (Nature, Vol. 355, pp. 379-380, (1992)) describes theuse of mixed micelles comprising a drug agent and biological lipids.

Similarly, micelles have also been used to deliver medications topatients, (Brodin et al., Acta Pharm. Suec. 19 267-284 (1982)) andmicelles have been used as drug carriers and for targeted drug delivery,(Supersaxo et al., Pharm. Res. 8:1286-1291 (1991)), including cancermedications, (Fung et al., Biomater. Artif. Cells. Artif. Organs 16: 439et. seq. (1988); and Yokoyama et al., Cancer Res. 51: 3229-3236 (1991)).

Hydrophobic agents of great interest include the polypyrrolic macrocyclebased photosensitizing compounds and, in particular green porphyrinssuch as BPD-MA (benzoporphyrin derivative monoacid ring A, also know byits generic name, verteporfin). These compounds have been known for sometime to be useful, when combined with light, for the treatment anddiagnosis of a variety of conditions, including tumors, angiogenesis andneovasculature, restenosis and atherosclerotic plaques, and rheumatoidarthritis. The porphyrins have a natural tendency to “localize” inmalignant or proliferating tissue, where they absorb light at certainwavelengths when irradiated. The absorbed light may result in acytotoxic effect in the cells, and neighboring cells, into which theporphyrins have localized. (See, e.g., Diamond et al., Lancet, 2:1175-77(1972); Dougherty et al., “The Science of Photo Medicine”, 625-38 (Reganet al. eds. 1982); and Dougherty et al., “Cancer: Principles andPractice of Oncology”, 1836-44 (DeVita Jr. et al. eds. 1982)). It hasbeen postulated that the cytotoxic effect of porphyrins is due to theformation of singlet oxygen when exposed to light (Weishaupt et al.,Cancer Research, 36:2326-29 (1976)).

Accordingly, preparations containing the porphyrins are useful in thediagnosis and the detection of important cells and tissue (see, e.g.“Porphyrin Photosensitization”, Plenum Press (Kessel et al. eds. 1983)),such as those related to tumors, growing vasculature, arterial blockageand autoimmunity. Similar photosensitizers have been used in thedetection and treatment of atherosclerotic plaques, as disclosed in U.S.Pat. Nos. 4,512,762 and 4,577,636. In addition to systemic use for thediagnosis and treatment of various conditions, the porphyrins can beused in a variety of other therapeutic applications. Porphyrin compoundshave been used topically to treat various skin diseases, as disclosed inU.S. Pat. No. 4,753,958.

A number of porphyrin photosensitizer preparations have been disclosedfor therapeutic applications. A photosensitizer preparation widely usedduring the early days of photodynamic therapy both for detection andtreatment was a crude derivative of hematoporphyrin, also calledhematoporphyrin derivative (“HPD”) or Lipson derivative, prepared asdescribed by Lipson et al., J. Natl. Cancer Inst., 26:1-8 (1961). Apurified form of the active component(s) of HPD was prepared byDougherty and co-workers by adjustment of the pH to cause aggregation,followed by recovery of the aggregate, as disclosed in U.S. Pat. Nos.4,649,151; 4,866,168; 4,889,129; and 4,932,934. A purified form of thisproduct is being used clinically under the trademark Photofrin® (AxcanPharmaceuticals), which is porfimer sodium.

Of particular interest is a group of modified porphyrins, known as“green porphyrins” (Gp), having one or more light absorption maximabetween about 670-780 nm. These Gp compounds have been shown to confercytotoxicity against target cells at concentrations lower than thoserequired for hematoporphyrin or HPD. Gp compounds can be obtained usingDiels-Alder reactions of protoporphyrin with various acetylenederivatives under the appropriate conditions. Preferred forms of Gp arethe hydro-monobenzoporphyrin derivatives (“BPD's”) as well as BPD-MA,EA6 and B3 in particular. The preparation and use of the Gp and BPDcompounds are disclosed in U.S. Pat. Nos. 4,920,143, 4,883,790 and5,095,030, hereby incorporated by reference into the disclosure of thepresent application. The preparation and uses of EA6 and B3 aredisclosed in U.S. Pat. Nos. 6,153,639 and 5,990,149 respectively, alsohereby incorporated by reference.

Many desirable hydro-monobenzoporphyrin photosensitizers, such asBPD-MA, are not only insoluble in water at physiological pH's, but arealso insoluble in (1) pharmaceutically acceptable aqueous-organicco-solvents, (2) aqueous polymeric solutions, and (3)surfactant/micellar solutions. It has recently been shown that theencapsulation of certain drugs in liposomes, prior to administration,has a marked effect on the pharmacokinetics, tissue distribution,metabolism and efficacy of the therapeutic agent. In an effort toincrease the tumor selectivity of porphyrin photosensitizers, porphyrincompounds have been incorporated into unilamellar liposomes, resultingin a larger accumulation and a more prolonged retention of thephotosensitizer by both cultured malignant cells and in experimentaltumors in vivo. Jori et al., Br. J. Cancer, 48:307-309 (1983); Cozzaniet al., In Porphyrins in Tumor Phototherapy, 177-183, Plenum Press(Andreoni et al. eds. 1984). This more efficient targeting of tumortissues by liposome-associated porphyrins may be due in part to thespecific delivery of phospholipid vesicles to serum lipoproteins, whichhave been shown to interact preferentially with hyperproliferativetissue, such as tumors, through receptor-mediated endocytosis. In thismanner, the selectivity of porphyrin uptake by tumors has beenincreased, as compared with photosensitizers dissolved in aqueoussolution. See Zhou et al., Photochemistry and Photobiology, 48:487-92(1988).

Accordingly, hematoporphyrin and hematoporphyrin dimethyl esters havebeen formulated in unilamellar vesicles of dipalmitoyl phosphatidylcholine (DPPC) and liposomes of dimyristoyl (DMPC) and distearoylphosphatidyl choline (DSPC). Zhou et al., supra; Ricchelli, NewDirections in Photodynamic Therapy, 847:101-106 (1987); Milanesi, Int.J. Radiat. Biol., 55:59-69 (1989). Similarly, HP, porfimer sodium, andtetrabenzoporphyrins have been formulated in liposomes composed of eggphosphatidyl choline (EPC). Johnson et al., Proc. Photodynamic TherapyMechanisms II, Proc. SPIE-Int. Soc. Opt. Eng., 1203:266-80 (1990).Additionally, BPD-MA can be “solubilized” at a concentration of about2.0 mg/ml in aqueous solution using an appropriate mixture ofphospholipids to form encapsulating liposomes. Such “solubilized”liposome compositions are suitable for parenteral administration.

Further, freeze-dried pharmaceutical formulations comprising a porphyrinphotosensitizer, a disaccharide or polysaccharide, and one or morephospholipids (such as EPG and DMPC) have been made. These formulationsform liposomes containing an effective amount of porphyrinphotosensitizer upon reconstitution with a suitable aqueous vehicle andare described in Desai et al., U.S. Pat. No. 6,074,666, which isincorporated by reference. Methods for the large-scale production ofDMPC/EPG liposomes containing a photosensitizer are disclosed in U.S.Pat. No. 5,707,608, which is incorporated by reference as if fully setforth.

It has been a challenge to find suitable pharmaceutical formulations forhydrophobic polypyrrolic macrocyle based photosensitizers that can befilter sterilized and freeze dried, and can also be rapidlyreconstituted in an aqueous medium prior to administration, whileretaining a small particle size after rehydration. Photosensitivecompounds such as verteporfin (BPD-MA) and QLT 0074 (EA6) must belyophilized for storage, because they are labile in an aqueousenvironment.

SUMMARY OF THE INVENTION

The present invention provides a phospholipid composition into whichhydrophobic photosensitizers may be incorporated that could be processedinto a stable liposome product small enough to be sterile filtered,lyophilized for storage, and would rapidly dissolve in an aqueous mediumfor administration, while maintaining the small particle size. It wasinitially believed that the phospholipids of choice would contain onlysaturated lipids, because saturated lipids are more stable, eliminatingthe need for anti-oxidants in pharamaceutical preparation. The initialattempts for a composition using saturated phospholipids failed.Surprisingly, it was found that the presence of at least someunsaturated lipid in the composition was essential for a stable, robustproduct that would survive the lyophilization process intact.Additionally, it was found that the presence of at least somephospholipids having negatively charged polar headgroups contributed tothe stability of the composition.

Another totally unexpected finding was that bilayer formingphospholipids comprising a proportion of unsaturated charged lipids werecapable of assuming a micellular structure (with or without theincorporation of a hydrophobic molecule) if the material was subjectedto a high energy process, such as microfluidization. The production ofmicelles from bilayer forming lipids is believed to be completely novel,and would not have been predicted from the literature on bilayer forminglipids.

The present invention relates to microaggregates of lipids andhydrophobic agents. In particular, the microaggregates are produced bycombining phospholipids and active hydrophobic compounds. Suchcompositions may be used in any therapeutic, agricultural or industrialsetting, and as such, they are delivery vehicles for the activehydrophobic agents. Preferably, the microaggregates comprise micellesand/or small liposomes containing a therapeutically acceptable amount ofa hydro-monobenzoporphyrin photosensitizer. The lipids used formicroaggregate production comprise unsaturated lipids, and may bestabilized by the presence of antioxidants. Preferably, themicroaggregates comprise a mixture of saturated and unsaturated lipids.Preferably, the microaggregates comprise phospholipids having aheadgroup that is negatively charged over the pH range of 5-7.Alternatively, the microaggregates may comprise both micelles andliposomes produced from, or containing, the same combination ofphospholipids.

The present invention also relates to methods of producingmicroaggregates comprising lipids and hydrophobic agents. It has beendiscovered that with appropriate selection of lipids, salt conditions,temperature, and size reduction process, microaggregates comprisingdiffering amounts of liposomes and micelles can be produced.Appropriately selected combinations of lipids, low salt conditions, anda high energy process such as microfluidization can result in theproduction predominantly micelle comprising microaggregate compositions.

The microaggregates of the invention provide nearly 100% incorporationof a hydrophobic agent such as a hydro-monobenzoporphyrinphotosensitizer, which can be expensive and usually requires acomplicated synthetic procedure to produce. Thus, there is littlereworking necessary and very little waste of the photosensitizer. Inaddition, due to their small particle size, the present microaggregatesexhibit the improved filterability important in producing largequantities of photosensitizer-containing delivery vehicles. Further, themicroaggregates retain their small size following lyophilization andreconstituion with an aqueous medium for pharmaceutical delivery. Suchphotosensitizing microaggregate compositions are useful in mediating thedestruction of unwanted cells or tissues or other undesirable materials,or to detect their presence through fluorescence, upon appropriateirradiation. Particularly preferred hydro-monobenzoporphyrinphotosensitizers used in the practice of this invention include thosehaving one or more light absorption maxima in the range of 670-780 nm.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects, features, and advantages are evident from the followingdescriptions of the various embodiments and the accompanying drawings,in which:

The FIGURE is a graphic representation of ³¹P-NMR of liposomes andmicelles in the presence of Mn²⁺.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to water soluble microaggregates (MA) ofhydrophobic agents and phospholipids or lipids. Water solublemicroaggregates are those which are miscible in water or other aqueoussolutions. Microaggregates refer to submicron size aggregates of regularor irregular, and spherical or non-spherical shape. For aggregates ofroughly spherical shape, the approximate diameters are less than onemicrometer. For significantly non-spherical aggregates, the approximatediameter of the aggregate when rotating is less than one micrometer.Aggregates refer to compositions comprising any aggregated complex ofconstituent molecules. Hydrophobic agents refer to those which arepoorly soluble (less than 5 mg/ml water) or insoluble in water or otheraqueous solutions.

Hydrophobic agents for formulation into the MA of the invention includeany that may be used pharmaceutically, agriculturally or industrially.These include biologically active, or otherwise useful, molecules,pharmaceuticals, imaging agents, and manufacturing reagents as well asprecursors and prodrugs of such substances. Preferred hydrophobic agentsare those with biological activity or other utility in humans and otherliving organisms. These include agents that are therapeutics inmedicine, ingredients in cosmetics, and pesticides and herbicides inagriculture. Examples of such agents include agonists and antagonists,analgesic and anti-inflammatory agents, anesthetics, antiadrenergic andantarrhythmics, antibiotics, anticholinergic and cholinomimetic agents,anticonvulsant agents, antidepressants, anti-epileptics, antifungal andantiviral agents, antihypertensive agents, antimuscarinic and muscarinicagents, antineoplastic agents, antipsychotic agents, anxiolytics,hormones, hypnotics and sedatives, immunosuppressive and immunoactiveagents, neuroleptic agents, neuron blocking agents, and nutrients.Particularly preferred agents include porphyrin photosensitizers such as“green porphyrins” such as BPD-MA, EA6 and B3. Generally, anypolypyrrolic macrocyclic photosensitive compound that is hydrophobic canbe used in the invention.

Examples of these and other photosensitizers for use in the presentinvention include, but are not limited to, angelicins, some biologicalmacromolecules such as lipofuscin; photosystem II reaction centers; andD1-D2-cyt b-559 photosystem II reaction centers, chalcogenapyrilliumdyes, chlorins, chlorophylls, coumarins, cyanines, ceratin DNA andrelated compounds such as adenosine; cytosine;2′-deoxyguanosine-5′-monophosphate; deoxyribonucleic acid; guanine;4-thiouridine; 2′-thymidine 5′-monophosphate;thymidylyl(3′-5′)-2′-deoxyadenosine;thymidylyl(3′-5′)-2′-deoxyguanosine; thymine; and uracil, certain drugssuch as adriamycin; afloqualone; amodiaquine dihydrochloride;chloroquine diphosphate; chlorpromazine hydrochloride; daunomycin;daunomycinone; 5-iminodaunomycin; doxycycline; furosemide; gilvocarcinM; gilvocarcin V; hydroxychloroquine sulfate; lumidoxycycline;mefloquine hydrochloride; mequitazine; merbromin (mercurochrome);primaquine diphosphate; quinacrine dihydrochloride; quinine sulfate; andtetracycline hydrochloride, certain flavins and related compounds suchas alloxazine; flavin mononucleotide; 3-hydroxyflavone; limichrome;limitlavin; 6-methylalloxazine; 7-methylalloxazine; 8-methylalloxazine;9-methylalloxazine; 1-methyl limichrome; methyl-2-methoxybenzoate;5-nitrosalicyclic acid; proflavine; and riboflavin, fullerenes,metalloporphyrins, metallophthalocyanines, methylene blue derivatives,naphthalimides, naphthalocyanines, certain natural compounds such asbis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione;4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one; N-formylkynurenine;kynurenic acid; kynurenine; 3-hydroxykynurenine; DL-3-hydroxykynurenine;sanguinarine; berberine; carmane; and 5,7,9(11),22-ergostatetraene-3β-ol, nile blue derivatives, NSAIDs (nonsteroidal anti-inflammatorydrugs), perylenequinones, phenols, pheophorbides, pheophytins,photosensitizer dimers and conjugates, phthalocyanines, porphycenes,porphyrins, psoralens, purpurins, quinones, retinoids, rhodamines,thiophenes, verdins, vitamins and xanthene dyes (Redmond and Gamlin,Photochem. Photobiol., 70(4):391-475 (1999)).

Exemplary angelicins include 3-aceto-angelicin; angelicin; 3,4′-dimethylangelicin; 4,4′-dimethyl angelicin; 4,5′-dimethyl angelicin;6,4′-dimethyl angelicin; 6,4-dimethyl angelicin; 4,4′,5′-trimethylangelicin; 4,4′,5′-trimethyl-1′-thioangelicin;4,6,4′-trimethyl-1′-thioangelicin; 4,6,4′-trimethyl angelicin;4,6,5′-trimethyl-1′-thioangelicin; 6,4,4′-trimethyl angelicin;6,4′,5′-trimethyl angelicin; 4,6,4′,5′-tetramethyl-1′-thioangelicin; and4,6,4′,5′-tetramethyl angelicin.

Exemplary chalcogenapyrillium dyes include pyrilium perchlorate,4,4′-(1,3-propenyl)-bis[2,6-di(1,1-dimethylethyl)]-; pyriliumperchlorate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)selenopyran-4-ylidene]-3-propenyl-;pyrilium hexofluoro phosphate,2,6-bis-(1,1-dimethyl-ethyl)-selenopyran-4-ylidene]-3-propenyl-;pyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-selenopyran-4-ylidene]-3-propenyl-; pyriliumperchlorate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-4-ylidene]-3-propenyl-;pyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-4-ylidene]-3-propenyl-;pyrilium perchlorate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)thiapyran-4-ylidene]-3-propenyl]-;selenopyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)selenopyran-4-ylidene]-3-propenyl]-;selenopyrilium,2,6-bis(1,1-dimethylethyl)-4-[1-[2,6-bis(1,1-dimethylethyl)selenopyran-4-ylidene]-3-propenyl]-;selenopyrilium percheorate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-4-ylidene]-3-propenyl]-;selenopyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-4-ylidene]-3-propenyl]-;selenopyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[2-[2,6-bis(1,1-dimethyl-ethyl)selenopyran-4-ylidene]-4-(2-butenyl)]-;selenopyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[2-[2,6-bis(1,1-dimethyl-ethyl)selenopyran-4-ylidene]-4-(2-pentenyl)]-;telluropyrilium tetrafluoroborate,2,6-bis(1,1-dimethylethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)-telluropyran-4-ylidene]-3-propenyl]-;telluropyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-4-ylidene]-3-propenyl]-;telluropyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-4-ylidene]ethyl-;telluropyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)-telluropyran-4-ylidene]methyl-;thiopyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)thiopyran-4-ylidene]-3-propenyl]-;thiopyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)selenopyran-4-ylidene]-3-propenyl]-;and thiopyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-4-ylidene]-3-propenyl]-.

Exemplary chlorins dyes include 5-azachlorin dimethyl ester derivative;5,10,15,20-tetrakis-(m-hydroxyphenyl)bacteriochlorin; benzoporphyrinderivative monoacid ring A; benzoporphyrin derivative monoacid ring-A;porphine-2,18-dipropanoic acid,7-[2-dimethyl-amino)-2-oxoethyl]-8-ethylidene-7,8-dihydro-3,7,12,17-tetramethyl,dimethylester; porphine-2,18-dipropanoic acid,7-[2-dimethyl-amino)-2-oxoethyl]-8-ethylidene-8-ethyl-7,8-dihydro-3,7,12,17-tetramethyl,dimethylester Z; porphine-2,18-dipropanoic acid,7-[2-dimethyl-amino)-2-oxoethyl]-8-ethylidene-8-ethyl-7,8-dihydro-3,7,12,17-tetramethyl,dimethylester Z ECHL; porphine-2,18-dipropanoic acid,7-[2-dimethyl-amino)-2-oxoethyl]-8-ethylidene-8-n-heptyl-7,8-dihydro-3,7,12,17-tetramethyl,dimethylester Z; tin (II) porphine-2,18-dipropanoic acid,7-[2-(dimethylamino-2-oxoethyl]-8-ethylidene-8-n-heptyl-7,8-dihydro-3,7,12,17-tetramethyl,dimethylester Z; chlorin e₆; chlorin e₆ dimethyl ester; chlorin e₆ k₃;chlorin e₆ monomethyl ester; chlorin e₆ Na₃; chlorin p₆; chlorinp₆-trimethylester; chlorin derivative zinc (II)porphine-2,18-dipropanoic acid,7-[2-(dimethylamino)-2-oxoethyl]-8-ethylidene-8-n-heptyl-7,8-dihydro-3,7,12,17-tetramethyl,dimethylester Z; 13¹-deoxy-20-formyl-vic-dihydroxy-bacteriochlorindi-tert-butyl aspartate; 13¹-deoxy-20-formyl-4-keto-bacteriochlorindi-tert-butyl aspartate; di-L-aspartyl chlorin e₆; mesochlorin;5,10,15,20-tetrakis-(m-hydroxyphenyl)chlorin;meta-(tetrahydroxyphenyl)chlorin;methyl-13¹-deoxy-20-formyl-4-keto-bacteriochlorin; mono-L-aspartylchlorin e₆; photoprotoporphyrin IX dimethyl ester; phycocyanobilindimethyl ester; protochlorophyllide a; tin (IV) chlorin e₆; tin chlorine₆; tin L-aspartyl chlorin e₆; tin octaethyl-benzochlorin; tin (IV)chlorin; zinc chlorin e₆; and zinc L-aspartyl chlorin e₆.

Exemplary chlorophylls dyes include chlorophyll a; chlorophyll b; oilsoluble chlorophyll; bacteriochlorophyll a; bacteriochlorophyll b;bacteriochlorophyll c; bacteriochlorophyll d; protochlorophyll;protochlorophyll a; amphiphilic chlorophyll derivative 1; andamphiphilic chlorophyll derivative 2.

Exemplary coumarins include 3-benzoyl-7-methoxycoumarin;7-diethylamino-3-thenoylcoumarin; 5,7-dimethoxy-3-(1-naphthoyl)coumarin;6-methylcoumarin; 2H-selenolo[3,2-g] [1] benzopyran-2-one;2H-selenolo[3,2-g] [1] benzothiopyran-2-one; 7H-selenolo[3,2-g] [1]benzoseleno-pyran-7-one; 7H-selenopyrano[3,2-f] [1] benzofuran-7-one;7H-selenopyrano[3,2-f] [1] benzo-thiophene-7-one; 2H-thienol[3,2-g] [1]benzopyran-2-one; 7H-thienol[3,2-g] [1] benzothiopyran-7-one;7H-thiopyrano[3,2-f] [1] benzofuran-7-one; coal tar mixture; khellin; RG708; RG277; and visnagin.

Exemplary cyanines include benzoselenazole dye; benzoxazole dye;1,1′-diethyloxacarbocyanine; 1,1′-diethyloxadicarbocyanine;1,1′-diethylthiacarbocyanine; 3,3′-dialkylthiacarbocyanines (n=2-18);3,3′-diethylthiacarbocyanine iodide; 3,3′-dihexylselenacarbocyanine;kryptocyanine; MC540 benzoxazole derivative; MC540 quinoline derivative;merocyanine 540; and meso-ethyl, 3,3′-dihexylselenacarbocyanine.

Exemplary fullerenes include C₆₀; C₇₀; C₇₆; dihydro-fullerene;1,9-(4-hydroxy-cyclohexano)-buckminster-fullerene;[1-methyl-succinate-4-methyl-cyclohexadiene-2,3]-buckminster-fullerene;and tetrahydro fullerene.

Exemplary metalloporphyrins include cadmium (II) chlorotexaphyrinnitrate; cadmium (II) meso-diphenyl tetrabenzoporphyrin; cadmiummeso-tetra-(4-N-methylpyridyl)-porphine; cadmium (II) texaphyrin;cadmium (II) texaphyrin nitrate; cobaltmeso-tetra-(4-N-methylpyridyl)-porphine; cobalt (II)meso(4-sulfonatophenyl)-porphine; copper hematoporphyrin; coppermeso-tetra-(4-N-methylpyridyl)-porphine; copper (II)meso(4-sulfonatophenyl)-porphine; Europium (III) dimethyltexaphyrindihydroxide; gallium tetraphenylporphyrin; ironmeso-tetra(4-N-methylpyridyl)-porphine; lutetium (III)tetra(N-methyl-3-pyridyl)-porphyrin chloride; magnesium (II)meso-diphenyl tetrabenzoporphyrin; magnesium tetrabenzoporphyrin;magnesium tetraphenylporphyrin; magnesium (II)meso(4-sulfonatophenyl)-porphine; magnesium (II) texaphyrin hydroxidemetalloporphyrin; magnesium meso-tetra-(4-N-methylpyridyl)-porphine;manganese meso-tetra-(4-N-methylpyridyl)-porphine; nickelmeso-tetra(4-N-methylpyridyl)-porphine; nickel (II)meso-tetra(4-sulfonatophenyl)-porphine; palladium (II)meso-tetra-(4-N-methylpyridyl)-porphine; palladiummeso-tetra-(4-N-methylpyridyl)-porphine; palladium tetraphenylporphyrin;palladium (II) meso(4-sulfonatophenyl)-porphine; platinum (II)meso(4-sulfonatophenyl)-porphine; samarium (II) dimethyltexaphyrindihydroxide; silver (II) meso(4-sulfonatophenyl)-porphine; tin (IV)protoporphyrin; tin meso-tetra-(4-N-methylpyridyl)-porphine; tinmeso-tetra(4-sulfonatophenyl)-porphine; tin (IV)tetrakis(4-sulfonatophenyl)porphyrin dichloride; zinc (II)15-aza-3,7,12,18-tetramethyl-porphyrinato-13,17-diyl-dipropionicacid-dimethylester; zinc (II) chlorotexaphyrin chloride; zinccoproporphyrin III; zinc (II)2,11,20,30-tetra-(1,1-dimethyl-ethyl)tetranaphtho(2,3-b:2′,3′-g:2″3″-1:2′″3′″-q)porphyrazine;zinc (II)2-(3-pyridyloxy)benzo[b]-10,19,28-tri(1,1-dimethylethyl)trinaphtho[2′,3′-g:2″3″1::2′″,3′″-q]porphyrazine;zinc (II)2,18-bis-(3-pyridyloxy)dibenzo[b,1]-10,26-di(1,1-dimethyl-ethyl)dinaphtho[2′,3′-g:2′″,3′″-q]porphyrazine;zinc (II)2,9-bis-(3-pyridyloxy)dibenzo[b,g]-17,26-di(1,1-dimethyl-ethyl)dinaphtho[2″,3″1:2′″,3′″-q]porphyrazine; zinc (II)2,9,16-tris-(3-pyridyloxy)tribenzo[b,g,1]-24=(1,1-dimethyl-ethyl)naphtho[2′″,3′″-q]porphyrazine;zinc (II)2,3-bis-(3-pyridyloxy)benzo[b]-10,19,28-tri(1.1-dimethyl-ethyl)trinaphtho[2′,3′-g:2″,3″1:2′″,3′″-q]porphyrazine;zinc (II)2,3,18,19-tetrakis-(3-pyridyloxy)dibenzo[b,1]-10,26-di(1,1-dimethyl-ethyl)trinaphtho[2′,3′-g:2′″,3′″-q]porphyrazine;zinc (II)2,3,9,10-tetrakis-(3-pyridyloxy)dibenzo[b,g]-17,26-di(1,1-dimethyl-ethyl)dinaphtho[2″,3″-1:2′″,3′″-q]porphyrazine;zinc (II)2,3,9,10,16,17-hexakis-(3-pyridyloxy)tribenzo[b,g,1]-24-(1,1-dimethyl-ethyl)naphtho[2′″,3′″-q]porphyrazine;zinc (II)2-(3-N-methyl)pyridyloxy)benzo[b]-10,19,28-tri(1,1-dimethyl-ethyl)trinaphtho[2′,3′-g:2″,3″1:2′″,3′″-q]porphyrazinemonoiodide; zinc (II)2,18-bis-(3-(N-methyl)pyridyloxy)dibenzo[b,1]-10,26-di(1,1-dimethylethyl)dinaphtho[2′,3′-g:2′″,3′″-q]porphyrazinediiodide; zinc (II)2,9-bis-(3-(N-methyl)pyridyloxy)dibenzo[b,g]-17,26-di(1,1-dimethylethyl)dinaphtho[2″,3″-1:2′″,3′″-q]porphyrazinediiodide; zinc (II)2,9,16-tris-(3-(N-methyl-pyridyloxy)tribenzo[b,g,1]-24-(1,1-dimethylethyl)naphtho[2′″,3′″-q]porphyrazinetriiodide; zinc (II)2,3-bis-(3-(N-methyl)pyridyloxy)benzo[b]-10,19,28-tri(1,1-dimethylethyl)trinaphtho[2′,3′-g:2″,3″-1:2′″,3′″-q]porphyrazinediiodide; zinc (II)2,3,18,19-tetrakis-(3-(N-methyl)pyridyloxy)dibenzo[b,1]-10,26-di(1,1-dimethyl)dinaphtho[2′,3′-g:2′″,3′″-q]porphyrazinetetraiodide; zinc (II)2,3,9,10-tetrakis-(3-(N-methyl)pyridyloxy)dibenzo[g,g]-17,26-di(1,1-dimethylethyl)dinaphtho[2″,3″-1:2′″,3′″-q]porphyrazinetetraiodide; zinc (II)2,3,9,10,16,17-hexakis-(3-(N-methyl)pyridyloxy)tribenzo[b,g,1]-24-(1,1-dimethylethyl)naphtho[2′″,3′″-q]porphyrazinehexaiodide; zinc (II) meso-diphenyl tetrabenzoporphyrin; zinc (II)meso-triphenyl tetrabenzoporphyrin; zinc (II)meso-tetrakis(2,6-dichloro-3-sulfonatophenyl)porphyrin; zinc (II)meso-tetra-(4-N-methylpyridyl)-porphine; zinc (II)5,10,15,20-meso-tetra(4-octyl-phenylpropynyl)-porphine; zinc porphyrinc; zinc protoporphyrin; zinc protoporphyrin IX; zinc (II)meso-triphenyl-tetrabenzoporphyrin; zinc tetrabenzoporphyrin; zinc (II)tetrabenzoporphyrin; zinc tetranaphthaloporphyrin; zinctetraphenylporphyrin; zinc (II) 5,10,15,20-tetraphenylporphyrin; zinc(II) meso (4-sulfonatophenyl)-porphine; and zinc (II) texaphyrinchloride.

Exemplary metallophthalocyanines include aluminummono-(6-carboxy-pentyl-amino-sulfonyl)-trisulfo-phthalocyanine; aluminumdi-(6-carboxy-pentyl-amino-sulfonyl)-trisulfophthalocyanine; aluminum(III) octa-n-butoxy phthalocyanine; aluminum phthalocyanine; aluminum(III) phthalocyanine disulfonate; aluminum phthalocyanine disulfonate;aluminum phthalocyanine disulfonate (cis isomer); aluminumphthalocyanine disulfonate (clinical prep.); aluminum phthalocyaninephthalimido-methyl sulfonate; aluminum phthalocyanine sulfonate;aluminum phthalocyanine trisulfonate; aluminum (III) phthalocyaninetrisulfonate; aluminum (III) phthalocyanine tetrasulfonate; aluminumphthalocyanine tetrasulfonate; chloroaluminum phthalocyanine;chloroaluminum phthalocyanine sulfonate; chloroaluminum phthalocyaninedisulfonate; chloroaluminum phthalocyanine tetrasulfonate;chloroaluminum-t-butyl-phthalocyanine; cobalt phthalocyanine sulfonate;copper phthalocyanine sulfonate; copper (II)tetra-carboxy-phthalocyanine; copper (II)-phthalocyanine; coppert-butyl-phthalocyanine; copper phthalocyanine sulfonate; copper (II)tetrakis-[methylene-thio[(dimethyl-amino)methylidyne]]phthalocyaninetetrachloride; dichlorosilicon phthalocyanine; gallium (III)octa-n-butoxy phthalocyanine; gallium (II) phthalocyanine disulfonate;gallium phthalocyanine disulfonate; gallium phthalocyaninetetrasulfonate-chloride; gallium (II) phthalocyanine tetrasulfonate;gallium phthalocyanine trisulfonate-chloride; gallium (II)phthalocyanine trisulfonate; GaPcS₁tBu₃; GaPcS₂tBu₂; GaPcS₃tBu₁;germanium (IV) octa-n-butoxy phthalocyanine; germanium phthalocyaninederivative; silicon phthalocyanine derivative; germanium (IV)phthalocyanine octakis-alkoxy-derivatives; iron phthalocyaninesulfonate; lead (II)2,3,9,10,16,17,23,24-octakis(3,6-dioxaheptyloxy)phthalocyanine;magnesium t-butyl-phthalocyanine; nickel (II)2,3,9,10,16,17,23,24-octakis(3,6-dioxaheptyloxy)phthalocyanine;palladium (II) octa-n-butoxy phthalocyanine; palladium (II)tetra(t-butyl)-phthalocyanine; (diol) (t-butyl)₃-phthalocyanatopalladium(II); ruthenium(II)dipotassium[bis(triphenyl-phosphine-monosulphonate)phthalocyanine;silicon phthalocyanine bis(tri-n-hexyl-siloxy)-; silicon phthalocyaninebis(tri-phenyl-siloxy)-; HOSiPcOSi(CH₃)₂(CH₂)₃N(CH₃)₂;HOSiPcOSi(CH₃)₂(CH₂)₃N(CH₂CH₃)₂; SiPc[OSi(CH₃)₂(CH₂)₃N(CH₃)₂]₂;SiPc[OSi(CH₃)₂(CH₂)₃N(CH₂CH₃)(CH₂)₂N(CH₃)₂]₂; tin (IV) octa-n-butoxyphthalocyanine; vanadium phthalocyanine sulfonate; zinc (II)octa-n-butoxy phthalocyanine; zinc (II)2,3,9,10,16,17,23,24-octakis(2-ethoxy-ethoxy)phthalocyanine; zinc (II)2,3,9,10,16,17,23,24-octakis(3,6-dioxaheptyloxy)phthalocyanine; zinc(II) 1,4,8,11,15,18,22,25-octa-n-butoxy-phthalocyanine;zn(II)-phthalocyanine-octabutoxy; zn(II)-phthalocyanine; zincphthalocyanine; zinc (II) phthalocyanine; zinc phthalocyanine andperdeuterated zinc phthalocyanine; zinc (II) phthalocyanine disulfonate;zinc phthalocyanine disulfonate; zinc phthalocyanine sulfonate; zincphthalocyanine tetrabromo-; zinc (II) phthalocyanine tetra-t-butyl-;zinc (II) phthalocyanine tetra-(t-butyl)-; zinc phthalocyaninetetracarboxy-; zinc phthalocyanine tetrachloro-; zinc phthalocyaninetetrahydroxyl; zinc phthalocyanine tetraiodo-; zinc ((I)tetrakis-(1,1-dimethyl-2-phthalimido)ethyl phthalocyanine; zinc (II)tetrakis-(1,1-dimethyl-2-amino)-ethyl-phthalocyanine; zinc (II)phthalocyanine tetrakis(1,1-dimethyl-2-trimethyl ammonium)ethyltetraiodide; zinc phthalocyanine tetrasulphonate; zinc phthalocyaninetetrasulfonate; zinc (II) phthalocyanine tetrasulfonate; zinc (II)phthalocyanine trisulfonate; zinc phthalocyanine trisulfonate; zinc (II)(t-butyl)₃-phthalocyanine diol; zinctetradibenzobarreleno-octabutoxy-phthalocyanine; zinc (II)2,9,16,23,-tetrakis-(3-(N-methyl)pyridyloxy)phthalocyanine tetraiodide;and zinc (II)2,3,9,10,16,17,23,24-octakis-(3-(N-methyl)pyridyloxy)phthalocyaninecomplex octaiodide; and zinc (II)2,3,9,10,16,17,23,24-octakis-(3-pyridyloxy)phthalocyanine.

Exemplary methylene blue derivatives include 1-methyl methylene blue;1,9-dimethyl methylene blue; methylene blue; methylene blue (16 μM);methylene blue (14 μM); methylene violet; bromomethylene violet;4-iodomethylene violet;1,9-dimethyl-3-dimethyl-amino-7-diethyl-amino-phenothiazine; and1,9-dimethyl-3-diethylamino-7-dibutyl-amino-phenothiazine.

Exemplary naphthalimides blue derivatives includeN,N′-bis-(hydroperoxy-2-methoxyethyl)-1,4,5,8-naphthaldiimide;N-(hydroperoxy-2-methoxyethyl)-1,8-naphthalimide; 1,8-naphthalimide;N,N′-bis(2,2-dimethoxyethyl)-1,4,5,8-naphthaldiimide; andN,N′-bis(2,2-dimethylpropyl)-1,4,5,8-naphthaldiimide.

Exemplary naphthalocyanines include aluminumt-butyl-chloronaphthalocyanine; siliconbis(dimethyloctadecylsiloxy)2,3-naphthalocyanine; siliconbis(dimethyloctadecylsiloxy) naphthalocyanine; siliconbis(dimethylthexylsiloxy)2,3-naphthalocyanine; siliconbis(dimethylthexylsiloxy)naphthalocyanine; siliconbis(t-butyldimethylsiloxy)2,3-naphthalocyanine; siliconbis(tert-butyldimethylsiloxy)naphthalocyanine; siliconbis(tri-n-hexylsiloxy) 2,3-naphthalocyanine; siliconbis(tri-n-hexylsiloxy)naphthalocyanine; silicon naphthalocyanine;t-butylnaphthalocyanine; zinc (II) naphthalocyanine; zinc (II)tetraacetyl-amidonaphthalocyanine; zinc (II) tetraminonaphthalocyanine;zinc (II) tetrabenzamidonaphthalocyanine; zinc (II)tetrahexylamidonaphthalocyanine; zinc (II)tetramethoxy-benzamidonaphthalocyanine; zinc (II)tetramethoxynaphthalocyanine; zinc naphthalocyanine tetrasulfonate; andzinc (II) tetradodecylamidonaphthalocyanine.

Exemplary nile blue derivatives include benzo[a]phenothiazinium,5-amino-9-diethylamino-; benzo[a]phenothiazinium,5-amino-9-diethylamino-6-iodo-; benzo[a]phenothiazinium,5-benzylamino-9-diethylamino-; benzo[a]phenoxazinium,5-amino-6,8-dibromo-9-ethylamino-; benzo[a]phenoxazinium,5-amino-6,8-diiodo-9-ethylamino-; benzo[a]phenoxazinium,5-amino-6-bromo-9-diethylamino-; benzo[a]phenoxazinium,5-amino-9-diethylamino-(nile blue A); benzo[a]phenoxazinium,5-amino-9-diethylamino-2,6-diiodo-; benzo[a]phenoxazinium,5-amino-9-diethylamino-2,-iodo; benzo[a]phenoxazinium,5-amino-9-diethylamino-6-iodo-; benzo[a]phenoxazinium,5-benzylamino-9-diethylamino-(nile blue 2B);5-ethylamino-9-diethylamino-benzo[a]phenoselenazinium chloride;5-ethylamino-9-diethyl-aminobenzo[a]phenothiazinium chloride; and5-ethylamino-9-diethyl-aminobenzo[a]phenoxazinium chloride.

Exemplary NSAIDs (nonsteroidal anti-inflammatory drugs) includebenoxaprofen; carprofen; carprofen dechlorinated(2-(2-carbazolyl)propionic acid); carprofen (3-chlorocarbazole);chlorobenoxaprofen; 2,4-dichlorobenoxaprofen; cinoxacin; ciprofloxacin;decarboxy-ketoprofen; decarboxy-suprofen; decarboxy-benoxaprofen;decarboxy-tiaprofenic acid; enoxacin; fleroxacin; fleroxacin-N-oxide;flumequine; indoprofen; ketoprofen; lomelfloxacin;2-methyl-4-oxo-2H-1,2-benzothiazine-1,1-dioxide; N-demethyl fleroxacin;nabumetone; nalidixic acid; naproxen; norfloxacin; ofloxacin;pefloxacin; pipemidic acid; piroxicam; suprofen; and tiaprofenic acid.

Exemplary perylenequinones include hypericins such as hypericin;hypericin monobasic sodium salt; di-aluminum hypericin; di-copperhypericin; gadolinium hypericin; terbium hypericin, hypocrellins such asacetoxy hypocrellin A; acetoxy hypocrellin B; acetoxy iso-hypocrellin A;acetoxy iso-hypocrellin B;3,10-bis[2-(2-aminoethylamino)ethanol]hypocrellin B;3,10-bis[2-(2-aminoethoxy)ethanol]hypocrellin B;3,10-bis[4-(2-aminoethyl)morpholine]hypocrellin B; n-butylaminatedhypocrellin B; 3,10-bis(butylamine) hypocrellin B;4,9-bis(butylamine)hypocrellin B; carboxylic acid hypocrellin B;cystamine-hypocrellin B; 5-chloro hypocrellin A or 8-chloro hypocrellinA; 5-chloro hypocrellin B or 8-chloro hypocrellin B; 8-chlorohypocrellin B; 8-chloro hypocrellin A or 5-chloro hypocrellin A;8-chloro hypocrellin B or 5-chloro hypocrellin B; deacetylated aldehydehypocrellin B; deacetylated hypocrellin B; deacetylated hypocrellin A;deacylated, aldehyde hypocrellin B; demethylated hypocrellin B;5,8-dibromo hypocrellin A; 5,8-dibromo hypocrellin B; 5,8-dibromoiso-hypocrellin B; 5,8-dibromo[1,12-CBr═CMeCBr(COMe)]hypocrellin B;5,8-dibromo[1,12-CHBrC(═CH₂)CBr(COMe)]hypocrellin B;5,8-dibromo[1-CH₂COMe, 12-COCOCH₂Br-]hypocrellin B; 5,8-dichlorohypocrellin A; 5,8-dichloro hypocrellin B; 5,8-dichlorodeacytylatedhypocrellin B; 5,8-diiodo hypocrellin A; 5,8-diiodo hypocrellin B;5,8-diiodo[1,12-CH═CMeCH(COCH₂I₂)-]hypocrellin B;5,8-diiodo[1,12-CH₂C(CH₂I)═C(COMe)-]hypocrellin B;2-(N,N-diethylamino)ethylaminated hypocrellin B;3,10-bis[2-(N,N-diethylamino)-ethylamine]hypocrellin B;4,9-bis[2-(N,N-diethyl-amino)-ethylamine] iso-hypocrellin B;dihydro-1,4-thiazine carboxylic acid hypocrellin B; dihydro-1,4-thiazinehypocrellin B; 2-(N,N-dimethylamino)propylamine hypocrellin B;dimethyl-1,3,5,8,10,12-hexamethoxy-4,9-perylenequinone-6,7-diacetate;dimethyl-5,8-dihydroxy-1,3,10,13-tetramethoxy-4,9-perylenequinone-6,7-diacetate;2,11-dione hypocrellin A; ethanolamine hypocrellin B; ethanolamineiso-hypocrellin B; ethylenediamine hypocrellin B; 11-hydroxy hypocrellinB or 2-hydroxy hypocrellin B; hypocrellin A; hypocrellin B;5-iodo[1,12-CH₂C(CH₂I)═C(COMe)-]hypocrellin B;8-iodo[1,12-CH₂C(CH₂I)═C(COMe)-]hypocrellin B; 9-methylaminoiso-hypocrellin B; 3,10-bis[2-(N,N-methylamino)propylamine]hypocrellinB; 4,9-bis(methylamine iso-hypocrellin B; 14-methylamine iso-hypocrellinB; 4-methylamine iso-hypocrellin B; methoxy hypocrellin A; methoxyhypocrellin B; methoxy iso-hypocrellin A; methoxy iso-hypocrellin B;methylamine hypocrellin B; 2-morpholino ethylaminated hypocrellin B;pentaacetoxy hypocrellin A; PQP derivative; tetraacetoxy hypocrellin B;5,8,15-tribromo hypocrellin B; calphostin C, Cercosporins such asacetoxy cercosporin; acetoxy iso-cercosporin; aminocercosporin;cercosporin; cercosporin+iso-cercosporin (1/1 molar);diaminocercosporin; dimethylcercosporin; 5,8-dithiophenol cercosporin;iso-cercosporin; methoxycercosporin; methoxy iso-cercosporin;methylcercosporin; noranhydrocercosporin; elsinochrome A; elsinochromeB; phleichrome; and rubellin A.

Exemplary phenols include 2-benzylphenol; 2,2′-dihydroxybiphenyl;2,5-dihydroxybiphenyl; 2-hydroxybiphenyl; 2-methoxybiphenyl; and4-hydroxybiphenyl.

Exemplary pheophorbides include pheophorbide a; methyl13¹-deoxy-20-formyl-7,8-vic-dihydro-bacterio-meso-pheophorbide a;methyl-2-(1-dodecyloxyethyl)-2-devinyl-pyropheophorbide a;methyl-2-(1-heptyl-oxyethyl)-2-devinyl-pyropheophorbide a;methyl-2-(1-hexyl-oxyethyl)-2-devinyl-pyropheophorbide a;methyl-2-(1-methoxy-ethyl)-2-devinyl-pyropheophorbide a;methyl-2-(1-pentyl-oxyethyl)-2-devinyl-pyropheophorbide a; magnesiummethyl bacteriopheophorbide d; methyl-bacteriopheophorbide d; andpheophorbide.

Exemplary pheophytins include bacteriopheophytin a; bacteriopheophytinb; bacteriopheophytin c; bacteriopheophytin d; 10-hydroxy pheophytin a;pheophytin; pheophytin a; and protopheophytin.

Exemplary photosensitizer dimers and conjugates include aluminummono-(6-carboxy-pentyl-amino-sulfonyl)-trisulfophthalocyanine bovineserum albumin conjugate; dihematoporphyrin ether (ester);dihematoporphyrin ether; dihematoporphyrin ether (ester)-chlorin;hematoporphyrin-chlorin ester; hematoporphyrin-low density lipoproteinconjugate; hematoporphyrin-high density lipoprotein conjugate;porphine-2,7,18-tripropanoic acid,13,13′-(1,3-propanediyl)bis[3,8,12,17-tetramethyl]-;porphine-2,7,18-tripropanoic acid,13,13′(1,11-undecanediyl)bis[3,8,12,17-tetramethyl]-;porphine-2,7,18-tripropanoic acid,13,13′(1,6-hexanediyl)bis[3,8,12,17-tetramethyl]-; SnCe6-MAb conjugate1.7:1; SnCe6-MAb conjugate 1.7:1; SnCe6-MAb conjugate 6.8:1; SnCe6-MAbconjugate 11.2:1; SnCe6-MAb conjugate 18.9:1; SnCe6-dextran conjugate0.9:1; SnCe6-dextran conjugate 3.5:1; SnCe6-dextran conjugate 5.5:1;SnCe6-dextran conjugate 9.9:1; α-terthienyl-bovine serum albuminconjugate (12:1); α-terthienyl-bovine serum albumin conjugate (4:1); andtetraphenylporphine linked to 7-chloroquinoline.

Exemplary phthalocyanines include (diol) (t-butyl)-3-phthalocyanine;(t-butyl)₄-phthalocyanine;cis-octabutoxy-dibenzo-dinaphtho-porphyrazine;trans-octabutoxy-dibenzo-dinaphtho-porphyrazine;2,3,9,10,16,17,23,24-octakis2-ethoxyethoxy)phthalocyanine;2,3,9,10,16,17,23,24-octakis(3,6-dioxaheptyloxy)phthalocyanine;octa-n-butoxy phthalocyanine; phthalocyanine; phthalocyanine sulfonate;phthalocyanine tetrasulphonate; phthalocyanine tetrasulfonate;t-butyl-phthalocyanine; tetra-t-butyl phthalocyanine; andtetradibenzobarreleno-octabutoxy-phthalocyanine.

Exemplary porphycenes include 2,3-(2³-carboxy-2⁴-methoxycarbonylbenzo)-7,12,17-tris(2-methoxyethyl)porphycene;2-(2-hydroxyethyl)-7,12,17-tri(2-methoxyethyl)porphycene;2-(2-hydroxyethyl)-7,12,17-tri-n-propyl-porphycene;2-(2-methoxyethyl)-7,12,17-tri-n-propyl-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-hydroxy-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-methoxy-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-n-hexyloxy-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-acetoxy-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-caproyloxy-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-pelargonyloxy-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-stearoyloxy-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-(N-t-butoxycarbonylglycinoxy)porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-[4-((β-apo-7-carotenyl)benzoyloxyl-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-amino-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-acetamido-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-glutaramido-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-(methyl-glutaramido)-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-(glutarimido)-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-3-(N,N-dimethylaminomethyl)-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-3-(N,N-dimethylaminomethyl)-porphycenehydrochloride; 2,7,12,17-tetrakis(2-ethoxyethyl)-porphycene;2,7,12,17-tetra-n-propyl-porphycene;2,7,12,17-tetra-n-propyl-9-hydroxy-porphycene;2,7,12,17-tetra-n-propyl-9-methoxy-porphycene;2,7,12,17-tetra-n-propyl-9-acetoxy porphycene;2,7,12,17-tetra-n-propyl-9-(t-butyl glutaroxy)-porphycene;2,7,12,17-tetra-n-propyl-9-(N-t-butoxycarbonylglycinoxy)-porphycene;2,7,12,17-tetra-n-propyl-9-(4-N-t-butoxy-carbonyl-butyroxy)-porphycene;2,7,12,17-tetra-n-propyl-9-amino-porphycene;2,7,12,17-tetra-n-propyl-9-acetamido-porphycene;2,7,12,17-tetra-n-propyl-9-glutaramido-porphycene;2,7,12,17-tetra-n-propyl-9-(methyl glutaramido)-porphycene;2,7,12,17-tetra-n-propyl-3-(N,N-dimethylaminomethyl)porphycene;2,7,12,17-tetra-n-propyl-9,10-benzo porphycene;2,7,12,17-tetra-n-propyl-9-p-benzoyl carboxy-porphycene;2,7,12,17-tetra-n-propyl-porphycene;2,7,12,17-tetra-t-butyl-3,6;13,16-dibenzo-porphycene;2,7-bis(2-hydroxyethyl)-12,17-di-n-propyl-porphycene;2,7-bis(2-methoxyethyl)-12,17-di-n-propyl-porphycene; and porphycene.

Exemplary porphyrins include 5-azaprotoporphyrin dimethylester;bis-porphyrin; coproporphyrin III; coproporphyrin III tetramethylester;deuteroporphyrin; deuteroporphyrin IX dimethylester;diformyldeuteroporphyrin IX dimethylester; dodecaphenylporphyrin;hematoporphyrin; hematoporphyrin (8 μM); hematoporphyrin (400 μM);hematoporphyrin (3 μM); hematoporphyrin (18 μM); hematoporphyrin (30μM); hematoporphyrin (67 μM); hematoporphyrin (150 μM); hematoporphyrinIX; hematoporphyrin monomer; hematoporphyrin dimer; hematoporphyrinderivative; hematoporphyrin derivative (6 μM); hematoporphyrinderivative (200 μM); hematoporphyrin derivative A (20 μM);hematoporphyrin IX dihydrochloride; hematoporphyrin dihydrochloride;hematoporphyrin IX dimethylester; haematoporphyrin IX dimethylester;mesoporphyrin dimethylester; mesoporphyrin IX dimethylester;monoformyl-monovinyl-deuteroporphyrin IX dimethylester;monohydroxyethylvinyl deuteroporphyrin;5,10,15,20-tetra(o-hydroxyphenyl)porphyrin;5,10,15,20-tetra(m-hydroxyphenyl)porphyrin;5,10,15,20-tetrakis-(m-hydroxyphenyl)porphyrin;5,10,15,20-tetra(p-hydroxyphenyl)porphyrin;5,10,15,20-tetrakis(3-methoxyphenyl)porphyrin;5,10,15,20-tetrakis(3,4-dimethoxyphenyl)porphyrin;5,10,15,20-tetrakis(3,5-dimethoxyphenyl) porphyrin;5,10,15,20-tetrakis(3,4,5-trimethoxyphenyl)porphyrin;2,3,7,8,12,13,17,18-octaethyl-5,10,15,20-tetraphenylporphyrin;Photofrin®; Photofrin® II; porphyrin c; protoporphyrin; protoporphyrinIX; protoporphyrin dimethylester; protoporphyrin IX dimethylester;protoporphyrin propylaminoethylformamide iodide; protoporphyrinN,N-dimethylaminopropylformamide; protoporphyrinpropylaminopropylformamide iodide; protoporphyrin butylformamide;protoporphyrin N,N-dimethylamino-formamide; protoporphyrin formamide;sapphyrin 1 3,12,13,22-tetraethyl-2,7,18,23 tetramethylsapphyrin-8,17-dipropanol; sapphyrin 2 3,12,13,22-tetraethyl-2,7,18,23tetramethyl sapphyrin-8-monoglycoside; sapphyrin 3;meso-tetra-(4-N-carboxyphenyl)-porphine;tetra-(3-methoxyphenyl)-porphine;tetra-(3-methoxy-2,4-difluorophenyl)-porphine;5,10,15,20-tetrakis(4-N-methylpyridyl)porphine;meso-tetra-(4-N-methylpyridyl)-porphine tetrachloride;meso-tetra(4-N-methylpyridyl)-porphine;meso-tetra-(3-N-methylpyridyl)-porphine;meso-tetra-(2-N-methylpyridyl)-porphine;tetra(4-N,N,N-trimethylanilinium)porphine;meso-tetra-(4-N,N,N″-trimethylamino-phenyl)porphine tetrachloride;tetranaphthaloporphyrin; 5,10,15,20-tetraphenylporphyrin;tetraphenylporphyrin; meso-tetra-(4-N-sulfonatophenyl)-porphine;tetraphenylporphine tetrasulfonate;meso-tetra(4-sulfonatophenyl)porphine; tetra(4-sulfonatophenyl)porphine;tetraphenylporphyrin sulfonate; meso-tetra(4-sulfonatophenyl)porphine;tetrakis(4-sulfonatophenyl)porphyrin;meso-tetra(4-sulfonatophenyl)porphine; meso(4-sulfonatophenyl)porphine;meso-tetra(4-sulfonatophenyl)porphine;tetrakis(4-sulfonatophenyl)porphyrin;meso-tetra(4-N-trimethylanilinium)-porphine; uroporphyrin; uroporphyrinI (17 μM); uroporphyrin IX; and uroporphyrin I (18 μM).

Exemplary psoralens include psoralen; 5-methoxypsoralen;8-methoxypsoralen; 5,8-dimethoxypsoralen; 3-carbethoxypsoralen;3-carbethoxy-pseudopsoralen; 8-hydroxypsoralen; pseudopsoralen;4,5′,8-trimethylpsoralen; allopsoralen; 3-aceto-allopsoralen;4,7-dimethyl-allopsoralen; 4,7,4′-trimethyl-allopsoralen;4,7,5′-trimethyl-allopsoralen; isopseudopsoralen;3-acetoisopseudopsoralen; 4,5′-dimethyl-isopseudopsoralen;5′,7-dimethyl-isopseudopsoralen; pseudoisopsoralen;3-acetopseudoisopsoralen; 3/4′,5′-trimethyl-aza-psoralen;4,4′,8-trimethyl-5′-amino-methylpsoralen;4,4′,8-trimethyl-phthalamyl-psoralen; 4,5′,8-trimethyl-4′-aminomethylpsoralen; 4,5′,8-trimethyl-bromopsoralen; 5-nitro-8-methoxy-psoralen;5′-acetyl-4,8-dimethyl-psoralen; 5′-aceto-8-methyl-psoralen; and5′-aceto-4,8-dimethyl-psoralen. Exemplary purpurins includeoctaethylpurpurin; octaethylpurpurin zinc; oxidized octaethylpurpurin;reduced octaethylpurpurin; reduced octaethylpurpurin tin; purpurin 18;purpurin-18; purpurin-18-methyl ester; purpurin; tin ethyl etiopurpurinI; Zn(II) aetio-purpurin ethyl ester; and zinc etiopurpurin.

Exemplary quinones include 1-amino-4,5-dimethoxy anthraquinone;1,5-diamino-4,8-dimethoxy anthraquinone; 1,8-diamino-4,5-dimethoxyanthraquinone; 2,5-diamino-1,8-dihydroxy anthraquinone;2,7-diamino-1,8-dihydroxy anthraquinone; 4,5-diamino-1,8-dihydroxyanthraquinone; mono-methylated 4,5- or 2,7-diamino-1,8-dihydroxyanthraquinone; anthralin (keto form); anthralin; anthralin anion;1,8-dihydroxy anthraquinone; 1,8-dihydroxy anthraquinone (Chrysazin);1,2-dihydroxy anthraquinone; 1,2-dihydroxy anthraquinone (Alizarin);1,4-dihydroxy anthraquinone (Quinizarin); 2,6-dihydroxy anthraquinone;2,6-dihydroxy anthraquinone (Anthraflavin); 1-hydroxy anthraquinone(Erythroxy-anthraquinone); 2-hydroxy-anthraquinone;1,2,5,8-tetra-hydroxy anthraquinone (Quinalizarin);3-methyl-1,6,8-trihydroxy anthraquinone (Emodin); anthraquinone;anthraquinone-2-sulfonic acid; benzoquinone; tetramethyl benzoquinone;hydroquinone; chlorohydroquinone; resorcinol; and 4-chlororesorcinol.

Exemplary retinoids include all-trans retinal; C₁₇ aldehyde; C₂₂aldehyde; 11-cis retinal; 13-cis retinal; retinal; and retinalpalmitate.

Exemplary rhodamines include 4,5-dibromo-rhodamine methyl ester;4,5-dibromo-rhodamine n-butyl ester; rhodamine 101 methyl ester;rhodamine 123; rhodamine 6G; rhodamine 6G hexyl ester;tetrabromo-rhodamine 123; and tetramethyl-rhodamine ethyl ester.

Exemplary thiophenes include terthiophenes such as2,2′:5′,2″-terthiophene; 2,2′:5′,2″-terthiophene-5-carboxamide;2,2′:5′,2″-terthiophene-5-carboxylic acid;2,2′:5′,2″-terthiophene-5-L-serine ethyl ester;2,2′:5′,2″-terthiophene-5-N-isopropynyl-formamide;5-acetoxymethyl-2,2′:5′,2″-terthiophene;5-benzyl-2,2′:5′,2″-terthiophene-sulphide;5-benzyl-2,2′:5′,2″-terthiophene-sulfoxide;5-benzyl-2,2′:5′,2″-terthiophene-sulphone;5-bromo-2,2′:5′,2″-terthiophene;5-(butynyl-3′″-hydroxy)-2,2′:5′,2″-terthiophene;5-carboxyl-5″-trimethylsilyl-2,2′:5′,2″-terthiophene;5-cyano-2,2′:5′,2″-terthiophene; 5,5″-dibromo-2,2′:5′,2″-terthiophene;5-(1′″,1′″-dibromoethenyl)-2,2′:5′,2″-terthiophene;5,5″-dicyano-2,2′:5′,2″-terthiophene;5,5″-diformyl-2,2′:5′,2″-terthiophene;5-difluoromethyl-2,2′:5′,2″-terthiophene;5,5″-diiodo-2,2′:5′,2″-terthiophene;3,3″-dimethyl-2,2′:5′,2″-terthiophene;5,5″-dimethyl-2,2′:5′,2″-terthiophene;5-(3′″,3′″-dimethylacryloyloxymethyl)-2,2′:5′,2″-terthiophene;5,5″-di-(t-butyl)-2,2′:5′,2″-terthiophene;5,5″-dithiomethyl-2,2′:5′,2″-terthiophene;3′-ethoxy-2,2′:5′,2″-terthiophene; ethyl2,2′:5′,2″-terthiophene-5-carboxylic acid;5-formyl-2,2′:5′,2″-terthiophene;5-hydroxyethyl-2,2′:5′,2″-terthiophene;5-hydroxymethyl-2,2′:5′,2″-terthiophene; 5-iodo-2,2′:5′,2″-terthiophene;5-methoxy-2,2′:5′,2″-terthiophene; 3′-methoxy-2,2′:5′,2″-terthiophene;5-methyl-2,2′:5′,2″-terthiophene;5-(3′″-methyl-2′″-butenyl)-2,2′:5′,2″-terthiophene; methyl2,2′:5′,2″-terthiophene-5-[3′″-acrylate]; methyl2,2′:5′,2″-terthiophene-5-(3′″-propionate);N-allyl-2,2′:5′,2″-terthiophene-5-sulphonamide;N-benzyl-2,2′:5′,2″-terthiophene-5-sulphonamide;N-butyl-2,2′:5′,2″-terthiophene-5-sulphonamide;N,N-diethyl-2,2′:5′,2″-terthiophene-5-sulphonamide;3,3′,4′,3″-tetramethyl-2,2′:5′,2″-terthiophene;5-t-butyl-5″-trimethylsilyl-2,2′:5′,2″-terthiophene;3′-thiomethyl-2,2′:5′,2″-terthiophene;5-thiomethyl-2,2′:5′,2″-terthiophene;5-trimethylsilyl-2,2′:5′,2″-terthiophene, bithiophenes such as2,2′-bithiophene; 5-cyano-2,2′-bithiophene; 5-formyl-2,2′-bithiophene;5-phenyl-2,2′-bithiophene; 5-(propynyl)-2,2′-bithiophene;5-(hexynyl)-2,2′-bithiophene; 5-(octynyl)-2,2′-bithiophene;5-(butynyl-4″-hydroxy)-2,2′-bithiophene;5-(pentynyl-5″-hydroxy)-2,2′-bithiophene;5-(3″,4″-dihydroxybutynyl)-2,2′-bithiophene derivative;5-(ethoxybutynyl)-2,2′-bithiophene derivative, and misclaneousthiophenes such as 2,5-diphenylthiophene; 2,5-di(2-thienyl)furan;pyridine,2,6-bis(2-thienyl)-; pyridine, 2,6-bis(thienyl)-; thiophene,2-(1-naphthalenyl)-; thiophene, 2-(2-naphthalenyl)-; thiophene,2,2′-(1,2-phenylene)bis-; thiophene, 2,2′-(1,3-phenylene)bis-;thiophene, 2,2′-(1,4-phenylene)bis-; 2,2′:5′,2″: 5″,2′″-quaterthiophene;α-quaterthienyl; α-tetrathiophene; α-pentathiophene; α-hexathiophene;and α-heptathiophene.

Exemplary verdins include copro (II) verdin trimethyl ester;deuteroverdin methyl ester; mesoverdin methyl ester; and zincmethylpyroverdin.

Exemplary vitamins include ergosterol (provitamin D2); hexamethyl-Co aCo b-dicyano-7-de(carboxymethyl)-7,8-didehydro-cobyrinate(Pyrocobester); pyrocobester; and vitamin D3.

Exemplary xanthene dyes include Eosin B(4′,5′-dibromo,2′,7′-dinitro-fluorescein, dianion); eosin Y; eosin Y(2′,4′,5′,7′-tetrabromo-fluorescein, dianion); eosin(2′,4′,5′,7′-tetrabromo-fluorescein, dianion); eosin(2′,4′,5′,7′-tetrabromo-fluorescein, dianion)methyl ester; eosin(2′,4′,5′,7′-tetrabromo-fluorescein, monoanion)p-isopropylbenzyl ester;eosin derivative (2′,7′-dibromo-fluorescein, dianion); eosin derivative(4′,5′-dibromo-fluorescein, dianion); eosin derivative(2′,7′-dichloro-fluorescein, dianion); eosin derivative(4′,5′-dichloro-fluorescein, dianion); eosin derivative(2′,7′-diiodo-fluorescein, dianion); eosin derivative(4′,5′-diiodo-fluorescein, dianion); eosin derivative(tribromo-fluorescein, dianion); eosin derivative(2′,4′,5′,7′-tetrachloro-fluorescein, dianion); eosin; eosindicetylpyridinium chloride ion pair; erythrosin B(2′,4′,5′,7′-tetraiodo-fluorescein, dianion); erythrosin; erythrosindianion; erythrosin B; fluorescein; fluorescein dianion; phloxin B(2′,4′,5′,7′-tetrabromo-3,4,5,6-tetrachloro-fluorescein, dianion);phloxin B (tetrachloro-tetrabromo-fluorescein); phloxine B; rose bengal(3,4,5,6-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein, dianion); rosebengal; rose bengal dianion; rose bengal O-methyl-methylester; rosebengal 6′-O-acetyl ethyl ester; rose bengal benzyl esterdiphenyl-diiodonium salt; rose bengal benzyl ester triethylammoniumsalt; rose bengal benzyl ester, 2,4,6,-triphenylpyrilium salt; rosebengal benzyl ester, benzyltriphenyl-phosphonium salt; rose bengalbenzyl ester, benzyltriphenyl phosphonium salt; rose bengal benzylester, diphenyl-iodonium salt; rose bengal benzyl ester,diphenyl-methylsulfonium salt; rose bengal benzyl ester,diphenyl-methyl-sulfonium salt; rose bengal benzyl ester,triethyl-ammonium salt; rose bengal benzyl ester, triphenyl pyrilium;rose bengal bis(triethyl-ammonium) salt)(3,4,5,6-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein,bis(triethyl-ammonium salt); rose bengal bis(triethyl-ammonium) salt;rose bengal bis(benzyl-triphenyl-phosphonium) salt(3,4,5,6-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein,bis(benzyl-triphenyl-phosphonium) salt); rose bengalbis(diphenyl-iodonium) salt(3,4,5,6-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein,bis(diphenyl-iodonium) salt); rose bengal di-cetyl-pyridinium chlorideion pair; rose bengal ethyl ester triethyl ammonium salt; rose bengalethyl ester triethyl ammonium salt; rose bengal ethyl ester; rose bengalmethyl ester; rose bengal octyl ester tri-n-butyl-ammonium salt RB; rosebengal, 6′-O-acetyl-, and ethyl ester.

Particularly preferred photosensitizers are the green porphyrins, suchas BPD-DA, -DB, -MA, and -MB, and in particular BPD-MA, EA6, and B3.These compounds are porphyrin derivatives obtained by reacting aporphyrin nucleus with an alkyne in a Diels-Alder type reaction toobtain a monohydrobenzoporphyrin, and they are described in detail inthe issued U.S. Pat. No. 5,171,749, which is hereby incorporated in itsentirety by reference. Of course, combinations of photosensitizers mayalso be used. It is preferred that the absorption spectrum of thephotosensitizer be in the visible range, typically between 350 nm and1200 nm, more preferably between 400-900 nm, and even more preferablybetween 600-900 nm.

BPD-MA is described, for example, in U.S. Pat. No. 5,171,749; EA6 and B3are described in U.S. Ser. Nos. 09/088,524 and 08/918,840, respectively,all of which are incorporated herein by reference. Preferred greenporphyrins have the basic structure:

where R⁴ is vinyl or 1-hydroxyethyl and R¹, R², and R³ are H or alkyl orsubstituted alkyl.

BPD-MA has the structure shown in formula 1 wherein R¹ and R² aremethyl, R⁴ is vinyl and one of R³ is H and the other is methyl. EA6 isof formula 2 wherein R¹ and R² are methyl and both R³ are 2-hydroxyethyl(i.e., the ethylene glycol esters). B3 is of formula 2 wherein R¹ ismethyl, R² is H, and both R³ are methyl. In both EA6 and B3, R⁴ is alsovinyl.

The representations of BPD-MA_(C) and BPD-MA_(D), which are thecomponents of Verteporfin, as well as illustrations of A and B ringforms of EA6 and B3, are as follows:

Related compounds of formulas 3 and 4 are also useful; in general, R⁴will be vinyl or 1-hydroxyethyl and R¹, R², and R³ are H or alkyl orsubstituted alkyl.

Microaggregates

The MA of the invention results in the production of phospholipidcontaining micelles, liposomes, and mixtures thereof. Phospholipidssuitable for use in the invention may be any naturally occurring orsynthetic phospholipid, whether saturated or unsaturated. They include,but not limited to, the following: phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidylglycerol, phosphatidic acid, lysophospholipids, egg orsoybean phospholipid or combinations thereof. The phospholipids may bein any form, including salted or desalted, hydrogenated or partiallyhydrogenated, or natural, semisynthetic (modified) or synthetic. Inpreferred embodiments of the invention, the phospholipids used are thosecapable of forming liposomes, but also able to result in the productionof micelles if a high energy processing step is used for size reductionof multilammelar liposomes.

Even more preferred are unsaturated phosphatidylglycerols orphosphatidylcholines with charged head groups. Such preferredembodiments include the use of negatively charged mono- orpolyunsaturated phosphatidylglycerols and phosphatidylcholines such asegg phosphatidylglycerol (EPG), palmitoyloleoylphosphatidylglycerol(POPG), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylcholine (DPPC), or combinations thereof. Theunsaturated fatty acid chain is preferably on the same phospholipidmolecule as the charged headgroup, but alternatively, the desiredcombination of unsaturation and charge could be attained by using acharged saturated molecule such as DMPG together with an unsaturatedphospholipid molecule. It will generally be preferable to limit theamount of the unsaturated phospholipid (in other words, not to make thewhole composition from unsaturated phospholipids) because of the greaterstability of saturated phospholipids. Preferably, the ratio ofunsaturated charged phospholipid to the saturated phospholipid is atleast about 1:99, and more preferably the ratio is at least about 3:97,and even more preferably in the range of about 10:90 or more. Mostpreferably, the ratio is in the range of about 40:60 to about 50:50, butmay exceed 50:50.

The number of unsaturations (double bonds) in the fatty acid chain canrange from about 1-6, but is more preferably about 1 to 3, and mostpreferably about 1 or about 2.

Without being bound by theory, and with respect to the preferential useof unsaturated lipids in the MA of the invention, it is believed thatsaturated acyl chains may not be sufficiently flexible duringlyophilization of (removing water from) the MA. Thus in the case ofliposomes, where water is removed from the core entrapped volume (forwhich an analogy of making raisins from grapes is applicable),unsaturated acyl chains permit more curvature in the lipid membrane andmay introduce the necessary flexibility to allow shrinkage duringdrying. As such, the micelle containing MA of the invention are lesssusceptible to these effects since they likely lack an inner water core(or alternatively have a significantly smaller one). This may explainthe robustness of micelle containing MA during lyophilization. Theflexibility of unsaturated lipids may be a likely cause of small stablemicelle structure formation during microfluidization. The presence ofunsaturated lipids also lowers the phase transition temperature (liquidto gel transition) of the formulation to below room temperature, andinduces a less pronounced transition. The amount of unsaturated lipiddetermines the degree to which the phase transition temperature isdecreased. It is also believed that the presence of a charged headgroupon a phospholipid (for example, on phosphotidylglycerol) stabilizessmall liposomes and micelles because the repulsive charge preventsfusion into larger liposomal structures.

All MA of the invention may comprise, consist of or consist essentiallyof any one or more phospholipids in combination with a hydrophobicagent. Preferably, the phospholipids used in the MA of the invention areeither synthetic or derived from non-animal sources. More preferably,the phospholipids used in the MA of the invention include DOPG (1,2dioleoylphosphatidylglycerol), which is a doubly unsaturated lipid ofplant origin.

Phosphatidyl glycerols (PGs) may also be present in the MA of theinvention. Examples of such PGs include dimyristoyl phosphatidylglycerol (DMPG), DLPG and the like. The incorporation of such PGs may beused to contribute to the stabilization of micelles. Other types ofsuitable lipids that may be included are phosphatidyl ethanolamines(PEs), phosphatidic acids (PAs), phosphatidyl serines, and phosphatidylinositols.

A range of total lipid to hydrophobic agent ratios may be use in thepractice of the invention. The ratio depends on the hydrophobic agentbeing used, but will assure the presence of a sufficient number of lipidmolecules to form stable MA. Appropriate total lipid:hydrophobic agentratios may be from about 7:1 and higher, although lower ratios also donot exhibit adverse effects. A preferred range is from about 7:1 to10:1. Of course all intermediate ratios within this range, such as about8:1 and about 9:1, are within the scope of the invention. Additionallywithin the scope of the invention are the sub-intermediate ratios withinthe range, such as from about 7.1:1 to 7.9:1, about 8.1:1 to 8.9:1, andabout 9.1:1 to 9.9:1, are within the scope of the invention. When thenumber of lipid molecules is not sufficient to form a stable complex,the lipophilic phase of the MA may become saturated with hydrophobicagent molecules. Then, any slight change in the process conditions canforce some of the previously encapsulated hydrophobic agent to leak outonto the surface of the MA, or even out into the aqueous phase.

If the concentration of hydrophobic agent is high enough, it canactually precipitate out from the aqueous layer and promote aggregationof the MA. The more unencapsulated hydrophobic agent present, the higherthe degree of aggregation. The more aggregation, the larger the meanaggregate size will be, and the MA will no longer be of a sufficientlysmall size for efficient use in steps such as filter sterilization. Thusslight increases in the lipid content can increase significantly thefilterability of the liposome composition by increasing the ability toform and maintain small aggregates. This is particularly advantageouswhen working with significant volumes of 500 ml, a liter, five liters,40 liters, or more, as opposed to smaller batches of about 100-500 ml orless.

When larger volumes of MA are being made, a higher molar ratio ofphospholipid provides more assurance of reliable aseptic filterabilityby providing smaller aggregates. Moreover, the substantial potencylosses that are common in scale-up batches, due at least in part tofilterability problems, can thus be avoided. Another means of increasingfilterability is by preparation of micelle containing MA since micellesare smaller than liposomes in general. Such micelle containing MA aremore readily filter sterilized with a 0.22 micron filter and a preferredembodiment of the invention. Additional advantages in MA containing thesmaller micelles is reduced loss of the active hydrophobic agent vialarge aggregates lost during filtering or other processes; and thestability of smaller aggregates after reconstitution. Thus a preferredembodiment of the invention is where the hydrophobic agent is present inamounts, or in ratios, that favor micelle formation.

When a combination of phospholipids is used in the MA of the invention,a range of relative lipid ratios may be used in combination with thetotal lipid:hydrophobic agent ratios described above. Appropriate lipidratios for combinations of two phospholipids range from about 50:50 toabout 97:1. Of course all intermediate ratios within this range, such asabout 70:30, about 80:20 and about 90:10, are within the scope of theinvention. As indicated by the use of the 99:1 ratio, sub-intermediateratios within the range, such as from about 71:29 to 79:21, about 81:19to 89:11, and about 91:9 to 97:3, are within the scope of the invention.Examples of combinations of two phospholipids where such ratios may beused include DMPC:DMPG, DMPC:EPG, DMPC:POPG and DMPC:DOPG. An additionalexample is DMPC:EPG, preferably at a ratio of about 5:3 respectively.With this combination, even higher hydrophobic agent:lipid ratios, suchas 1:10, 1:15, or 1:20, respectively, may be used.

A particularly preferred embodiment of the MA of the invention compriseshydrophobic agents in an 8:1 total phospholipid:hydrophobic agent ratiowith a 60:40 lipid ratio of a DMPC:DOPC combination containingantioxidants BHT and AP. In particular, hydrophobic agents such as EA6and/or BPD-MA may be used in such MA. Also preferred are MA compositionscomprising EA6 in small liposomes comprising lipids and other componentsas described herein.

Antioxidants

In preferred embodiments comprising the use of unsaturatedphospholipids, the invention encompasses the use of antioxidants toprevent oxidation of the phospholipids. Auto-oxidation of unsaturatedacyl chains has been known to be a problem for long-term storage ofliposome formulations. Failure to prevent oxidative breakdown ofunsaturated phospholipids results in subcomponents such as lyso lipidsand fatty acids, which may be undesirable in some MA compositions. Assuch, antioxidants suitable for inclusion in phospholipid containingmicroaggregates to improve long-term storage are known in the art.Examples of such antioxidants include butylated hydroxytoluene (BHT),alpha-tocopherol, and ascorbyl palmitate (AP) as well as pH bufferingagents such as phosphates and glycine. Preferably, BHT is present atabout 0.01-0.02% by weight and AP at about 0.1-0.2% by weight.

BHT is hydrophobic and would be expected to remain in the lipophilicenvironments of the MA of the invention. BHT has the ability to preventchain propagation during auto-oxidation by accepting radicals formedduring the oxidative breakdown of lipids. Ascorbic acid has the capacityto act as an antioxidant and to act with other antioxidants such asalpha-tocopherol. It has been shown that the BHT/ascorbic acid systemallows for BHT regeneration, following its conversion to a phenoxylradical after free radical scavenging from oxidized lipids, therebyresulting in the appearance of ascorbyl radicals. This latter factorjustifies the relative weight ration of AP to BHT described above. APwas used in place of ascorbic acid because the hydrophobic nature of theformer would be expected to concentrate the antioxidant withinlipophilic environments.

Another anti-oxidation considerations is the filling of containerheadspaces with nitrogen gas and the sealing of such containers.Additionally, and because metal ions can catalyze oxidative processes,the use of high quality drug, excipients, and containers, the judiciouscleaning of manufacturing equipment, and the appropriate use of metalion chelators are preferred.

Cryoprotective Agents and Isotonic Agents

In a preferred embodiment of the invention, the MA are stabilized bylyophilization. An advantage to the micelle containing MA of theinvention is the fact that micelles may be more readily lyophilized incomparison to liposomes due to the absence of a water core.Lyophilization of liposomes require the passage of water across at leastone lipid bilayer, resulting in increased processing times and expense.The absence of a water core also permits micelles to have a greaterconcentration of phospholipid per unit volume. Thus a larger amount ofhydrophobic agent can be solubilized by the phospholipid per unit volumeof micelle. This permits the final micelle MA delivery vehicle to have ahigher drug density per unit volume than other delivery vehicles, suchas liposomes alone.

MA of the invention may contain a cryoprotectant for stabilizing the MAduring lyophilization. Alternatively, the physical structures of the MAcan be preserved by the presence of sufficient water afterlyophilization. This is may be accomplished by appropriate control ofthe degree of lyophilization. Since there is no entrapped volume inmicelles, the micelle containing MA of the invention facilitates greatercontrol over water soluble components, like solvent or salt, to beremoved in the preparation of delivery vehicles requiring such removal.

Any cryoprotective agent known to be useful in the art of preparingfreeze-dried formulations, such as di- or polysaccharides or otherbulking agents such as lysine, may be used in the claimed invention.Further, isotonic agents typically added to maintain isomolarity withbody fluids may be used. In preferred embodiments, a di-saccharide orpolysaccharide is used and functions both as a cryoprotective agent andas an isotonic agent. In an especially preferred embodiment, thedisaccharide or polysaccharide is selected from among the groupconsisting of lactose, trehalose, maltose, maltotriose, palatinose,lactulose or sucrose, with lactose or trehalose being preferred.Effective sugars such as trehalose and lactose are capable of hydrogenbonding to the phospholipidhead group in place of water. It has alsobeen hypothesized that effective sugars also act a as a spacing matrixto decrease the opposition of phospholipids on the exterior of adjacentMA such as liposomes.

When the process of hydrating a lipid film is prolonged, largerliposomes tend to be formed, and hydrophobic agents may evenprecipitate. The addition of a disaccharide or polysaccharide providesthe largest surface area for depositing a thin film of MA and virtuallyinstantaneous subsequent hydration. This thin film provides for fasterhydration so that, when the MA are initially formed by adding theaqueous phase (hydrated), the MA are of a smaller and more uniformparticle size. This provides significant advantages in terms ofmanufacturing ease.

However, it is also possible that, when a saccharide is present in thecomposition of the invention, it is added after dry lipid filmformation, as a part of the aqueous solution used in hydration. In aparticularly preferred embodiment, a saccharide is added to the drylipid film of the invention during hydration.

Disaccharides or polysaccharides are preferred to monosaccharides forthis purpose. To keep the osmotic pressure of the MA compositions of theinvention similar to that of blood, no more than 4-5% monosaccharidesshould be added. In contrast, about 9-10% of a disaccharide can be usedwithout generating an unacceptable osmotic pressure. The higher amountof disaccharide provides for a larger surface area, which results insmaller particle sizes being formed during hydration of the lipid film.

Also, when present, the disaccharide or polysaccharide is formulated ina preferred ratio of about 10-20 saccharide to 0.5-6.0 totalphospholipids, respectively, even more preferably at a ratio from about10 to 1.5-4.0. In one embodiment, a preferred but not limitingformulation is lactose or trehalose and total phospholipids in a ratioof about 10 to 0.94-1.88 to about 0.65-1.30, respectively.

The presence of the disaccharide or polysaccharide in the compositionnot only tends to yield MA having extremely small and narrow aggregatesize ranges, but also provides MA compositions in which the hydrophobicagents, such as a hydro-monobenzoporphyrin photosensitizer, may bestably incorporated in an efficient manner, i.e., with an encapsulationefficiency approaching 80-100%. Moreover, MA made with a saccharidetypically exhibit improved physical and chemical stability, such thatthey can retain an incorporated hydrophobic agent, such ashydro-monobenzoporphyrin photosensitizer, without leakage upon prolongedstorage, either as a reconstituted aqueous suspension or as acryodesiccated powder.

Freeze-Drying

Once formulated, the MA of the invention may be freeze-dried forlong-term storage if desired. For example, BPD-MA, a preferredhydro-monobenzoporphyrin photosensitizer, has maintained its potency ina cryodesiccated MA composition for a period of at least nine months atroom temperature, and a shelf life of at least two years has beenprojected. If the composition is freeze-dried, it may be packed in vialsfor subsequent reconstitution with a suitable aqueous solution, such assterile water or sterile water containing a saccharide and/or othersuitable excipients, just prior to use. For example, reconstitution maybe by simply adding water for injection just prior to administration.

Various lyophilization techniques are known in the art. For example, MAcontaining vials of the invention may be first frozen to −45° C. andthen held there for a period of up to about 90 minutes. This may befollowed by a high vacuum primary drying cycle wherein the temperatureis increased slowly to up to about 10° C. for a period usually on theorder of about 50 hours. This may be followed by a 20° C. secondarydrying cycle of up to about 24 hours. Once the lyophilizer pressurestabilizes at about 55-65 mTorr (73-87 microbar), the cycle isterminated. Thereafter, the vials may be sealed after overlaying withnitrogen gas. A general rule for freeze-drying is that a solid, brittle,non-collapsed, and homogenous cake is preferred for successfulre-hydration.

Additionally, the use of lyophilization may prevent hydrolysis ofhydrophobic agents susceptible to such reactions. For example, thephotosensitizer BPD-MA may be hydrolyzed to BPD-DA.

Size

In one aspect of the invention, the MA are of a sufficiently small andnarrow size that the aseptic filtration of the composition through a0.22 micron hydrophilic filter can be accomplished efficiently and withlarge volumes of 500 ml to a liter or more without significant cloggingof the filter. As such micelle and small liposome containing MA are apreferred embodiment of the invention. Moreover, and given their smallersize, the MA of the invention may mainly, or predominantly, containhydrophobic agent bearing micelles. The MA of the invention may containgreater than about 50%, greater than about 60%, greater than about 75%,greater than about 80%, greater than about 90%, and greater than about95% micelles. Even more preferably, the MA of the invention may containgreater than about 97%, about 98%, or about 99% micelles. Mostpreferably in desired circumstances, the MA of the invention consistonly of micelles. Alternatively, the MA of the invention may in somecircumstances (when an extrusion process is used for size reduction ofmultilammelar liposomes, rather than a high energy process such asmicrofluidization) contain up to 100% liposomes.

Micelles refer to microaggregates with the hydrophobic (lipophilic)“tail” portion of the phospholipids generally oriented toward theinterior of the micelle. Preferably, micelles have the “tail” portiongenerally oriented toward the center of the micelle. Micelles do nothave a bilayer structure and so are not considered vesicles orliposomes. The micelles of the invention have average diameters of lessthan about 30 nm (nanometers). Preferably, they have average diametersof less than about 20 nm.

Liposomes refer to microaggregates comprising at least one phospholipidbilayer, composed of two lipid monolayers having a hydrophobic “tail”region and a hydrophilic “head” region. The structure of the membranebilayer is such that the hydrophobic (nonpolar) “tails” of the lipidmonolayers orient themselves towards the center of the bilayer, whilethe hydrophilic “heads” orient themselves toward the aqueous phase. Theygenerally comprise completely closed, lipid bilayer membranes thatcontain an entrapped aqueous volume. Given the bilayer structure, asignificant portion (up to about half) of the phospholipids will havetheir hydrophobic (lipophilic) portion generally oriented away from thecenter of the liposome. Liposomes include unilamellar vesicles having asingle membrane bilayer or multilamellar vesicles having multiplemembrane bilayers, each bilayer being separated from the next by anaqueous layer. The average diameters of liposomes are larger than thatof micelles.

In liposomes, a hydrophobic agent can be entrapped in the aqueous phaseof the liposome or be associated with the “tail” portion ofphospholipids in the lipid bilayer. In micelles, a hydrophobic agent isleft to associate only with the “tail” portion of phospholipids in thecore of the micelle. Additionally, both micelles and liposomes may beused to help “target” a hydrophobic drug to an active site or tosolubilize hydrophobic drugs for parenteral administration.

One aspect of the present invention uses this ability to form micellesand liposomes by the same mixture of hydrophobic agent andphospholipids. This would result in MA that have a bimodal distributionin their diameters, indicating the presence of both micelles andliposomes. In another aspect of the invention, the micelles andliposomes are form under conditions that favor one type ofmicroaggregate over the other in the same mixture. Conditions that favormicelle formation include the presence of low salt in the mixture aswell as the use of low salt aqueous solution for hydrating the driedmixture. “Low salt” refers to conditions containing less than about 0.1N free cations or anions. Preferably, it refers to less than about 0.01N free ions. More preferably it refers to less than about 0.001 N freeions.

Preferred MA of the invention have an average aggregate size diameter ofwell below about 300 nm, more preferably below from about 200 nm. Mostpreferably, the MA of the invention have an average aggregate sizediameter below about 100 nm, and sometimes, depending on the conditionschosen, in the range of 10-50 nm. The size of the microaggregates madecomprising QLT 0074, DOPG and DMPC (see Example 1 below) have been sizedusing three different methods (using a NICOMP 370 Submicron ParticleSizer, by freeze fracture analysis and by size exclusion HPLC). Freezefracture analysis showed a mixture of micelles (7-15 nm in diameter),and relatively few liposomes (between 6- and 270 nm diameter). Sizeexclusion HPLC indicated mean particle size of 28 nm when tested in fourdifferent media (PBS, 0.9% sodium chloride, 9.2% lactose and 5%dextrose) with a range or 25-35 nm.

As discussed herein, the invention controls four major parameters thatcan affect the ease of aggregate size reduction to an unexpected degree.As a result, the filterability, particularly with standard asepticfiltration, is significantly improved in the MA of the invention. Theseparameters are (1) the production of micelles and small liposomes by useof low salt conditions; (2) suitable molar ratio ofhydro-monobenzoporphyrin photosensitizer to total phospholipids; (3)temperature during the hydration step; and (4) temperature during thehomogenization or size reduction step. The latter two parameters arediscussed below.

Filterability can be tested by passing a MA composition through aMicrofluidizer™ three times and withdrawing a sample with a syringe. Thesyringe is connected to a 0.22 micron hydrophilic filter and then placedin a syringe pump. The constant rate of piston movement is set at 10ml/min, and filtrate is collected until the filter becomes blocked bylarge aggregates. The volume of the filtrate is then measured andrecorded in terms of ml/cm² or g/cm², with a square centimeter being theeffective filtration area. Thus, filterability for the purposes of theinvention is defined as the maximum volume or weight of MA compositionthat can be filtered through a 0.22 micron filter.

The MA of the invention may be used as a delivery vehicle for theconstituent hydrophobic agent to target any cell or tissue for whichcontact with the agent is desired. In preferred embodiments of theinvention, the agent is a photosensitizer to be delivered prior to lightirradiation as part of photodynamic therapy (PDT). Particularlypreferred MA of the invention comprise a hydro-monobenzoporphyrinphotosensitizer, including BPD-MA and EA6, for use in photodynamictherapy (PDT) or diagnosis.

The MA of the invention also preferably comprises micelles which arereadily, and significantly, destabilized in the presence of proteins,salts, charged elements, and/or polymers. Such MA are well suited as apharmaceutical formulation to deliver hydrophobic drugs to fluids suchas blood, which contains proteins, salts, charged elements and polymers.Given the ability to destabilize after delivery to target conditions,the MA of the invention can rapidly deliver hydrophobic agents totargets such as the bloodstream, where the drugs may be picked up ortransferred to blood components for further transport and/or targetingbased on the components' specificities. As such, the MA can beconsidered “fast breaking” in that the MA is stable in vitro but, whenadministered in vivo, the hydrophobic drug (such as a photosensitizer)is rapidly released into the bloodstream where it associates with bloodcomponents such as serum lipoproteins. Another beneficial effect of thistransfer is reduced depositing of hydrophobic agents in various organs,especially the liver. As such, the pharmokinetics of delivering thehydrophobic agent with such micelles are altered compared to the use ofother delivery vehicles or systems, such as those that do not releasethe agent rapidly or those that do not transfer the agent to bloodcomponents.

Preparation

Methods for the production of the MA of the invention comprise, consistof, and/or consisting essentially of the combination of hydrophobicagents and phospholipids and subjecting them to conditions capable offorming micelles, small liposomes or combinations thereof. Preferably,the methods comprise the use of phospholipids capable of forming lipidbilayers and result in the production of stable micelles and/or smallliposomes. The resultant MA, especially those comprising or consistingof micelles of the invention, do not contain detergents normally usedfor micelle production. The absence of detergents can markedly reducetoxicity known to result in hemolysis and kidney damage. To favormicelle formation, the MA of the invention are formulated under low saltconditions because, as noted above, the micelles of the invention aredestabilized by salt.

Generally, the MA of the invention are produced by dissolving thedesired MA constituent component molecules (such as desiredphospholipids, hydrophobic agent, and optionally antioxidants andcryoprotectants) into a solvent to form an “intermediate complex”.Preferred solvents are organic or otherwise non-aqueous. Suitableorganic solvents include any volatile organic solvent, such as diethylether, acetone, methylene chloride, chloroform, piperidine,piperidine-water mixtures, methanol, tert-butanol, dimethyl sulfoxide,N-methyl-2-pyrrolidone, and mixtures thereof. Preferably, the organicsolvent is water-immiscible, such as methylene chloride, but waterimmiscibility is not required. In any event, the solvent chosen shouldnot only be able to dissolve all of the components of the lipid film,but should also not react with, or otherwise deleteriously affect, thesecomponents to any significant degree.

The organic solvent is then removed from the resulting solution to forma dry lipid film by any known laboratory technique that is notsignificantly deleterious to the dry lipid film and the hydrophobicagent. Such techniques include any that remove the solvent via itsgaseous phase, including evaporation or vacuum. In one embodiment, thesolvent is removed by placing the solution under a vacuum until theorganic solvent is evaporated. The solid residue is the dry lipid filmof the invention, which contains aggregates of the MA components,considered the “presome”. The thickness of the lipid film is notcritical, but usually varies from about 30 to about 45 mg/cm², dependingupon the amount of solid residual and the surface area of the vesselwhich contains it. In another embodiment of the invention, the solventis removed as part the “presome” process of Nanba et al. (U.S. Pat. No.5,096,629, which is hereby incorporated by reference as if fully setforth), which heats the “intermediate complex” and subjects it todryness via an instantaneous vacuum drying system such as the CRUX 8B™(Orient Chemical Ind., Ltd., Japan) to produce a lipid powder containingaggregates of the MA components.

Once formed, the film or powder may be stored for an extended period oftime, preferably not more than 4 to 21 days, prior to hydration. Storagemay be under an appropriate gas, such as argon. While the temperatureduring a lipid film or powder storage period is also not an importantfactor, it is preferably below room temperature, most preferably in therange from about −20 to about 4° C. One advantage to the Nanba et al.“presome” process is the reduction of batch to batch variability seenwith thin film, which arises due to the use of multiple batches inevaporation vessels.

The dry lipid film or powder may be hydrated with an aqueous solution,preferably containing a disaccharide or polysaccharide if not previouslypresent. This will result in the formation of large multilammelarliposomes that can be further processed by extrusion or a high energyprocess, such as microfluidization to form the desired particle size.Examples of useful aqueous solutions used during the hydration stepinclude sterile water, or a dilute solution of lactose. In oneembodiment of the invention, the solution is physiologically isotonic,such as 9.2% lactose, which permits bolus injections. Preferably theaqueous solution is sterile. Most preferably for the production ofmicelles and the stabilization of small liposomes, the solution is lowsalt. It is believed that the presence of salts neutralizes the negativerepulsive charges that prevent the aggregation or fusion of these smallparticles into larger liposomes.

The volume of aqueous solution used during hydration can vary greatly,but should not be so great as about 98% nor so small as about 30-40%. Atypical range of useful volumes would be from about 50 or 60% to about95%, preferably about 75% to about 95%, more preferably about 80% toabout 90%, and most preferably about 85% to 90%. Of course all subrangesfrom about 30% to about 98% are included as part of the invention.

The physical manipulation of material during hydration may be conductedby a variety of means, including mixing and rotating on a rotaryevaporator, manual swirling of vessels, and the use of standardlaboratory stirrer or shaker means (including stir bars with stirplates, high shear mixers, paddles and combinations thereof). Preferredin the practice of the invention are high agitation methods, such as theuse of high-shear mixing or egg-shaped stir bars.

Upon hydration, coarse aggregates are formed that incorporate atherapeutically effective amount of the hydrophobic agent. The“therapeutically effective amount” can vary widely, depending on thetissue to be treated and whether the hydrophobic agent is coupled to atarget-specific ligand, such as an antibody or an immunologically activefragment. Typically, the therapeutically effective amount is such toproduce a dose of hydrophobic agent within a range of from about 0.1 toabout 20 mg/kg, preferably from about 0.15-2.0 mg/kg and, even morepreferably, from about 0.25 to about 0.75 mg/kg. Preferably, the w/vconcentration of the hydrophobic agent in the “intermediate complex”ranges from about 0.1 to about 8.0-10.0 g/L, when the mixture becomessuch a thick gel that it is not possible to handle or administer to asubject by the usual means. Most preferably, the concentration is about2.0 to 2.5 g/L.

It should be noted that if the agent is a photosensitizer, the variousparameters used for selective photodynamic therapy are interrelated.Therefore, the therapeutically effective amount should also be adjustedwith respect to other parameters, for example, fluence, irradiance,duration of the light used in photodynamic therapy, and the timeinterval between administration of the photosensitizing agent and thetherapeutic irradiation. Generally, all of these parameters are adjustedto produce significant damage to tissue deemed undesirable, such asneovascular or tumor tissue, without significant damage to thesurrounding tissue, or to enable the observation of such undesirabletissue without significant damage to the surrounding tissue.

The hydration step should take place at a temperature that does notexceed the glass transition temperature of the phospholipid andhydrophobic agent aggregates formed. For photosensitizers of theinvention, this temperature is about 30° C. Preferably the temperatureis at room temperature or lower, such as from 10-25, or even morepreferred from 15-20° C. or 17-22° C. An especially preferredtemperature is about 21° C. The glass transition temperature of thephospholipid and hydrophobic agent aggregates can be measured by using adifferential scanning microcalorimeter. Madden et al. (“Spontaneousvesiculation of large multilamellar vesicles composed of saturatedphosphatidylcholine and phosphatidylglycerol mixtures.” Biochemistry,Vol. 27, pp. 8724-8730, (1988)) describe the effects of temperature andionic strength on vesicle formation.

The use of unsaturated charged lipids as encompassed by the inventionmay effectively lower the phase transition temperature Tc (liquid to geltransition) of the formulation to below room temperature and induce aless pronounced transition. The amount of unsaturated lipid determinesthe degree of Tc lowering.

The particle sizes of the coarse aggregates first formed duringhydration are then homogenized to a more uniform size and/or reduced toa smaller size range of about less than about 50 to about 300 nm,depending on the method of size reduction used. Preferably, thishomogenization and/or reduction is also conducted at a temperature belowthe glass transition temperature of the hydrophobic agent-phospholipidcomplex formed in the hydration step. For photosensitizers of theinvention, such temperature does not exceed about 30° C., and ispreferably below room temperature of about 25° C. It has been found thatthe homogenization temperature with photosensitizers is preferably atroom temperature or lower, e.g., 15-20° C. At higher homogenizationtemperatures, such as about 32-42° C., the relative filterability of theMA composition may improve initially due to increased fluidity asexpected, but then, unexpectedly, tends to decrease with continuingagitation due to increasing particle size.

Various high-speed agitation or high energy system manipulationprocesses may be used during the homogenization step. Examples of suchprocesses include microfluidization (liquid jet milling), high shearmixing, and sonication. While effective, sonication is not ideal for usein large scale production of MA. Processing through the aforementionedhigh energy system results in the production of small particles, usuallya mixture of small liposomes and micelles. Extrusion, is another methodof size reduction. Extrusion results in the production of smallliposomes (as small as 50 to 100 nm), but micelles have not beenobserved by the inventors in production by this procedure. Extrusioninvolves the forcing of hydrated material, under pressure and attemperatures known to make liposome formulations fluid, through membranefilters of defined pore sizes. While adequate for laboratory scalebatches of material, extrusion may not be ideal for large scaleprocesses since 1) the pores become clogged even at high pressures ofgreater than 1000 psi, 2) the surface area of the filter membrane andextruder volume are limitations, and 3) multiple discontinuous passesthrough the extruder increases the likelihood of differences betweenbatches.

Devices for the above described processes include a Microfluidizer™(such as a Microfluidics™ Model 110F); a sonicator; a high-shear mixer;a homogenizer; a standard laboratory shaker or stirrer, or any otheragitation device. Of course modifications in such processes to suit theparticular hydrophobic agent of interest and formation of the desired MAare within the scope of the invention. In one preferred embodiment ofthe invention, these processes are used for the production of MAcontaining mainly micelles.

Such processes may be used to produce MA various ratios of micelles,liposomes and combinations thereof. In embodiments where both micellesand liposomes are produced, they may be separated by the bimodal sizedistribution seen in combinations of the two. This arises from thesignificantly smaller size of micelles in comparison to liposomes. Theanalysis of MA size may be performed by methods including electronmicroscopy, to exclude large aggregates as liposomes, and use of aparticle sizer, which may be used in combination with fitting routinesfor uni- and bimodal distributions. Another method is by use ofmanganese chloride (Mn²⁺) mediated nuclear magnetic resonance (³¹P-NMR),where ³¹Phosphorus labeled headgroups of lipids on the inner layer of aliposome lipid bilayer are not quenched by Mn²⁺ because Mn²⁺ cannotreadily cross the bilayer to enter the entrapped volume. Thus liposomeswill produce a residual NMR signal of about 30-40% for large and smallliposomes after adding Mn²⁺. All ³¹P-labeled headgroups of lipids of amicelle, however, are on the surface and thus fully exposed to Mn²⁺quenching. Thus micelles produce no remaining NMR signal due toquenching after adding Mn²⁺ (see the FIG.).

In a preferred embodiment, a high pressure device such as aMicrofluidizer™ is used for agitation. Some models of microfluidizationsystems are continuous and batch size scalable processors.Microfluidization uses high pressure streams of hydrated material thatcollide at ultra-high velocities in precisely defined microchannels. Inthe interaction chamber, two streams of fluid at a high speed collidewith each other at a 90° angle. The combined forces of shear, impact andcavitation result in the production of liposomes and micelles. Inmicrofluidization, a large amount of heat is generated during the shortperiod of time during which the fluid passes through a high pressureinteraction chamber. As the microfluidization temperature increases, thefluidity of the membrane also increases, which initially makes particlesize reduction easier, as expected. For example, filterability canincrease by as much as four times with the initial few passes through aMicrofluidizer™ device. The increase in the fluidity of the bilayermembrane promotes particle size reduction, which makes filtration of thefinal composition easier. In the initial several passes, this increasedfluidity mechanism advantageously dominates the process.

However, as the number of passes and the temperature both increase, moreof the hydrophobic agent molecules are apparently squeezed out in casesinvolving liposomes, increasing the tendency of the liposomes toaggregate into larger particles. At the point at which the aggregationof vesicles begins to dominate the process, the sizes cannot be reducedany further.

For this reason, in the methods of the invention, the homogenizationtemperature is cooled down to and maintained at a temperature no greaterthan room temperature after the composition passes through the zone ofmaximum agitation, e.g., the interaction chamber of a Microfluidizer™device. An appropriate cooling system can easily be provided for anystandard agitation device in which homogenization is to take place,e.g., a Microfluidizer™, such as by circulating cold water into anappropriate cooling jacket around the mixing chamber or other zone ofmaximum turbulence. While the pressure used in such high pressuredevices is not critical, pressures from about 10,000 to about 16,000 psiare not uncommon.

Maintaining the hydration temperature and the homogenizing/reducing stepat a temperature below 30° C. would not have been expected to producesmaller particle sizes. In fact, the invention is contrary to theconventional wisdom that small particle sizes are achieved by increasingrather than decreasing these temperatures. See, e.g., M. Lee et al.,“Size Distribution of Liposomes by Flow Field-Flow Fractionation”, J.Pharm. & Biomed. Analysis, 11:10, 911-20 (1993), equation (6) showingparticle diameter “d” as inversely related to temperature “T”, and FIG.6 b therein showing liposome preparation I (prepared at about 70° C.)having smaller particle sizes than preparation II (prepared at about 23°C.).

As a last step, the MA compositions of the inventions are preferablyaseptically filtered through a filter having an extremely small poresize, i.e., 0.22 micron. While other sterilization methods, such asheating and X-ray irradiation are known, in the art, the use of suchmethods may result in irreversible structural changes in lipids andhydrophobic agents such as many photosensitizers. A wide variety offiltration systems are known in the art, including Durapore TPcartridges, Millipak 100, Millidisk 40S, and millidisk MCGL. Filterpressures used during sterile filtration can vary widely, depending onthe volume of the composition, the density, the temperature, the type offilter, the filter pore size, and the size of the MA. However, as aguide, a typical set of filtration conditions would be as follows:filtration pressure of 15-25 psi; filtration load of 0.8 to 1.5 ml/cm²;and filtration temperature of about 25° C. Preferably, the hydrophilicMillidisk 40S is used at a load of approximately 1 ml/cm².

A typical general procedure for producing hydro-monobenzoporphyrinphotosensitizer containing MA of the invention is described below withadditional exemplary detail:

-   -   (1) Sterile filtration of methylene chloride as organic solvent        through a hydrophobic, 0.22 micron filter.    -   (2) Addition of DMPC:EPG:BPD-MA at a ratio of 4.7:3.25:1 and        excipients to the filtered organic solvent, dissolving both the        excipients and the photosensitizer to form the “intermediate        complex”.    -   (3) Filtration of the resulting solution through a 0.22 micron        hydrophobic filter.    -   (4) Transfer of the filtrate to a rotary evaporator apparatus,        such as that commercially available under the name        Rotoevaporator.    -   (5) Removal of the organic solvent to form a dry lipid film.    -   (6) Analysis of the lipid film to determine the level of organic        solvent concentration; optionally continuing removal until the        level of organic solvent is below 0.01%,    -   (7) Preparation of a 10% lactose solution. If the MA formulation        is to be injected, this solution should be injectable.    -   (8) Filtration of the lactose solution through a 0.22 micron        hydrophilic filter.    -   (9) Hydration of the lipid film with the filtered 10% lactose        solution to form coarse aggregates.    -   (10) Reduction of the particle sizes of the coarse aggregates by        passing them through a Microfluidizer™, optionally at 9000 psi        (pounds per square inch) for about 5 discrete passes to produce        micelles.    -   (11) Determination of the reduced aggregated size distribution        of MA.    -   (12) Aseptic filtration of the MA composition through a 0.22        micron hydrophilic filter. (Optionally, the solution may first        be pre-filtered with a 5.0 micron or smaller pre-filter.)    -   (13) Analysis of photosensitizer potency.    -   (14) Filling of vials with the MA composition.    -   (15) Freeze-drying.

The above may be adapted for the selective production of micelles byconducting all appropriate steps under low salt conditions to favorsubsequent micelle production after hydration. As such, salt basedbulking agents must not be used. In such applications, the resultingmicelles are on the order of about 15 nm in diameter, which is at thelower limit for feasible liposome sizes. The micelle structure wasconfirmed by use of ³¹P-NMR.

An alternative general procedure for producing hydro-monobenzoporphyrinphotosensitizer containing MA of the invention by use of a “presome”process of Nanba et al. (see U.S. Pat. No. 5,096,629) is described belowwith additional exemplary detail:

-   -   (1) Sterile filtration of methylene chloride as organic solvent        through a hydrophobic, 0.22 micron filter.    -   (2) Addition of DMPC:DOPG at a ratio of 60:40 with a total        lipid:EA6 at a ratio of 8:1 and antioxidants BHT and AP to the        filtered organic solvent, dissolving both the excipients and the        photosensitizer to form the “intermediate complex”.    -   (3) Filtration of the resulting solution through a 0.22 micron        hydrophobic filter.    -   (4) Transfer of the filtrate to liquid tank followed by feeding        to a tubular heater heated externally.    -   (5) Removal of the organic solvent by sending the heated mixture        into a vacuum chamber of no more than 300 mm Hg at a speed over        0.1 times the speed of sound to instantaneously dry the mixture        to form lipid powder.    -   (6) Analysis of the lipid powder to determine the level of        organic solvent concentration; optionally continuing removal        until the level of organic solvent is below 0.01%,    -   (7) Preparation of a 10% lactose solution. If the MA formulation        is to be injected, this solution should be injectable.    -   (8) Filtration of the lactose solution through a 0.22 micron        hydrophilic filter.    -   (9) Hydration of the lipid powder with the filtered 10% lactose        solution to form coarse aggregates.    -   (10) Dispersion of the coarse aggregates by stirring them at        high rpm at a temperature below the glass transition temperature        of the photosensitizer and phospholipid containing aggregates.    -   (11) Determination of the reduced aggregated size distribution        of MA.    -   (12) Aseptic filtration of the MA composition through a 0.22        micron hydrophilic filter. (Optionally, the solution may first        be pre-filtered with a 5.0 micron or smaller pre-filter.)    -   (13) Analysis of photosensitizer potency.    -   (14) Filling of vials with the MA composition.    -   (15) Freeze-drying.

One means of conducting the above instantaneous drying is by use of avacuum drying system such as the CRUX 8B™ product of Orient ChemicalInd., Ltd., Japan. Moreover, the above dispersion step may be at speedsof about 10,000 rpm, or ranging from 8000 to 15,000 rpm. Such a“presome” process may also be adapted for the selective production ofmicelles by conducting all appropriate steps under low salt conditionsto favor subsequent micelle production after hydration. As such, saltbased bulking agents must not be used.

As described above, the practice of the methods of the invention for MAproduction may be conducted with a variety of phospholipids andprocesses. The invention includes the observation, beyond the use of lowsalt conditions, that the use of charged, unsaturated phospholipids,such as EPG and DOPG, as well as high energy processing (such asmicrofluidization and sonication), appears to favor the formation ofmicelles in otherwise liposome forming combinations of phospholipids andhydrophobic agents. The use of unsaturated phospholipids provides anumber of desirable characteristics. These include the ability toconduct MA production steps at room temperature and to produce smallerMA when used in combination with saturated lipids.

Administration and Use

The use of the hydrophobic agents incorporated in the MA of theinvention may be for any appropriate pharmaceutical, agricultural orindustrial application. With incorporated photosensitizers, the MA maybe used for any condition or in any method for which thephotosensitizers are appropriate in combination with exposure to lightor other electromagnetic radiation. These include, but are not limitedto, the diagnosis or treatment of cancer, the reduction of activatedleukocytes, the treatment of ocular disorders, the treatment andprevention of neovasculature and angiogenesis, the destruction ofviruses and cells infected thereby, the treatment of atheroscleroticplaques, the treatment of restenosis, and others. In addition, manyphotosensitizers may be photoactivated by appropriate excitationwavelengths to fluoresce visibly. This fluorescence can then be used tolocalize a tumor or other target tissue. By incorporating hydrophobicagents in the MA of the invention, more efficient packaging, deliveryand hence administration of the agents can be obtained.

Generally speaking, the MA of the invention may be applied in any manneridentical or analogous to the administration of micelles and liposomes.The concentration of the hydrophobic agent in the MA of the inventiondepends upon the nature of the agent as well as the nature of theadministration desired. This dependency also exists in application ofhydro-monobenzoporphyrin photosensitizers via MA.

The MA compositions and formulations of the invention may beadministered parenterally or by injection. Injection may be intravenous,subcutaneous, intramuscular, intrathecal, or even intraperitoneal.However, the MA may also be administered by aerosol intranasally orintrapulmonarally, or topically. Formulations designed for timed releaseare also with the scope of the invention.

The quantity of hydrophobic agent MA formulation to be administereddepends on the choice of active agents, the conditions to be treated,the mode of administration, the individual subject, as well as theskill, experience and judgement of the practitioner. Generally speaking,however, dosages in the range of 0.05-10 mg/kg may be appropriate. Theforegoing range is, of course, merely suggestive, as the number ofvariables in regard to an individual treatment regime is large.Therefore, considerable excursions from these recommended values areexpected.

For example, and with the use of photosensitizers as a diagnostic inlocalizing tumor tissue or in localizing atherosclerotic plaques, the MAcompositions of the invention are administered systemically in the samegeneral manner as is known with respect to photodynamic therapy. Thewaiting period to allow the drugs to clear from tissues to which they donot accumulate is approximately the same, for example, from about 30minutes to about 10 hours. After the compositions of the invention havebeen permitted to localize, the location of the target tissue isdetermined by detecting the presence of the photosensitizer.

In diagnosis, the photosensitizers incorporated into MA may be usedalong with, or may be labeled with, a radioisotope or other detectingmeans. If this is the case, the detection means depends on the nature ofthe label. Scintigraphic labels such as technetium or indium can bedetected using ex vivo scanners. Specific fluorescent labels can also beused but, like detection based on fluorescence of the photosensitizersthemselves, these labels may require prior irradiation.

For activation of the photosensitizer applied by the MA of theinvention, any suitable absorption wavelength is used. This can besupplied using the various methods known to the art for mediatingcytotoxicity or fluorescence emission, such as visible radiation,including incandescent or fluorescent light sources or photodiodes suchas light emitting diodes. Laser light can also be used for in situdelivery of light to a localized photosensitizer. In a typical protocol,for example, several hours prior to irradiation, approximately 0.5-1.5mg/kg of green porphyrin photosensitizer containing MA is injectedintravenously and then excited by an appropriate wavelength.

The following example is presented to describe the preferredembodiments, utilities and attributes of the present invention, but theynot meant to limit the invention. The invention is not to be limited tothe particular photosensitizer used in the Example.

EXAMPLE 1 Production of QLT0074 for Injection

Five hundred mL methylene chloride was added to 0.001 g butylatedhydroxytoluene, 0.01 g ascorbyl palmitate, 3.2 g dioleoyl phosphatidylglycerol and 4.8 g dimyristoyl phosphatidyl choline in a pressure unitand mixed using an overhead stirrer until a clear solution was obtained.Once the solution was clear, 1 g QLT0074 crystals was slowly added underreduced light and mixed using an overhead stirrer until the crystalsdissolved completely. The solution was then filtered through a 0.22 μmfilter, and transferred to a round bottom flask. The flask was on arotary evaporator and the methylene chloride was removed under reducedpressure, with continued drying after the distillation stopped. Thevacuum was broken and the flask was attached to a vacuum manifold forfurther drying. Five hundred mL of sterile filtered 9.2% w/v lactosemonohydrate in water for injection was added to the QLT0074/lipid thinfilm and agitated at room temperature for at least 1 h to dissolve andproduce multilammelar vesicles. A Model M-1105 microfluidizer wasflushed with water then some lactose solution, and then theQLT0074/lipid solution until green solution appeared in the discharge.The following parameters were used: air pressure, 120 psi; operatingpressure, 10,030 psi; inlet air pressure gauge reading, 62 psi. Thecooling coil reservoir was filled with crushed ice and water to maintaina product temperature in a range of 16-20° C. The QLT/0074 lipidmaterial was processed 5 times through the microfluidizer. The resultingmaterial was then passed through 0.22 μm filters, and aliquoted intolabelled lyophilization vials, with 1 ml per aliquot. The material waslyophilized using a BCCA lyophilizer, Labconco, serial #215369. Thelyophilized samples were stored in the dark at 2-8° C.

All references cited herein, including patents, patent applications, andpublications, are hereby incorporated by reference in their entireties,whether previously specifically incorporated or not. As used herein, theterms “a”, “an”, and “any” are each intended to include both thesingular and plural forms.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

The invention claimed is:
 1. A lyophilized composition containing eggphosphatidylglycerol or dioleoyl phosphatidylglycerol (DOPG) asunsaturated phospholipids and dimyristoyl phosphatidylcholine (DMPC) assaturated phospholipids and one or more green porphyrinphotosensitizers, wherein the composition maintains its potency for aperiod of at least nine months at room temperature, and wherein thecomposition, upon rehydration, produces a composition comprisingmicelles with an average diameter below about 100 nm.
 2. The compositionof claim 1 wherein said composition contains at least one antioxidant.3. The composition of claim 2 wherein said at least one antioxidant isbutylated hydroxyl toluene (BHT) and/or ascorbyl palmitate (AP).
 4. Thecomposition of claim 1 wherein the unsaturated phospholipid is DOPG. 5.The composition of claim 1 wherein said one or more green porphyrinphotosensitizer is BPD-MA, BPD-DA, BPD-DB, EA6, B3 or a combinationthereof.
 6. The composition of claim 4 wherein the ratio of DOPG:DMPC is40:60.
 7. The composition of claim 1 wherein the ratio ofphospholipids:photosensitizer is 8:1.
 8. The composition of claim 1wherein the average diameter is below about 50 nm.
 9. The composition ofclaim 1 wherein the average diameter is below about 30 nm.
 10. Thecomposition of claim 1 wherein the average diameter is below about 20nm.
 11. A method for making a lyophilized composition which uponrehydration produces a composition comprising micelles with an averagediameter below about 100 nm containing one or more green porphyrinphotosensitizers and a mixture of saturated and unsaturatedphospholipids, wherein said method comprises: homogenizing at highenergy by microfluidization, sonication, or high speed shearing, at atemperature of 15-20° C., a composition of aggregates wherein saidcomposition of aggregates has been formed by hydrating a dried mixtureof said saturated and unsaturated phospholipids and green porphyrin toproduce by said homogenizing a composition containing micelles; andlyophilizing said composition comprising micelles to produce saidlyophilized composition, wherein said composition maintains its potencyfor a period of at least nine months at room temperature, and whereinsaid composition, upon rehydration, produces a composition comprisingmicelles with an average diameter below about 100 nm.
 12. The method ofclaim 11 wherein the unsaturated phospholipid is EPG or DOPG and thesaturated phospholipid is DMPC.
 13. The method of claim 11 wherein saidone or more green porphyrin photosensitizer is BPD-MA, BPD-DA, BPD-DB,EA6, B3 or a combination thereof.
 14. The method of claim 13 whereinsaid one or more photosensitizers is BPD-MA.
 15. The method of claim 12wherein said unsaturated lipid is DOPG.
 16. A method of providingphotodynamic therapy (PDT) to a subject, said method comprisingadministering a composition of claim 1, after rehydration, to saidsubject, and photoactivating the photosensitizer in said composition.17. A method of diagnosing the presence of cancer or localizingatherosclerotic plaques in a subject, said method comprisingadministering a composition of claim 1, after rehydration, to saidsubject and detecting the presence of the photosensitizer in a targettissue of said subject.
 18. The method of claim 16 wherein said one ormore photosensitizers comprises BPD-MA.
 19. A lyophilized compositionprepared by the method of claim
 11. 20. A lyophilized compositionprepared by the method of claim 12.